Inositol pyrophosphates, and methods of use thereof

ABSTRACT

The present invention comprises compounds, compositions thereof, and methods capable of delivering modified inositol hexaphospahte (IHP) comprising an internal pyrophosphate ring to the cytoplasm of mammalian cells. In certain embodiments, the present invention relates to compounds, compositions thereof, and methods that enhance the ability of mammalian red blood cells to deliver oxygen, by delivering IHP to the cytoplasm of the red blood cells.

RELATED APPLICATION INFORMATION

This application claims the benefit of priority under 35 U.S.C. section119(e) to Provisional Patent Applications 60/376,383, filed Apr. 29,2002; 60/388,851, filed Jun. 14, 2002; 60/395,749, filed Jul. 12, 2002;and 60/424,573, filed Nov. 7, 2002.

BACKGROUND OF THE INVENTION

I. Ischemia

Ischemic insult, i.e., the localized deficiency of oxygen to an organ orskeletal tissue, is a common and significant problem in many clinicalconditions. The problem is especially acute in organ transplantoperations in which a harvested organ is removed from a body, isolatedfrom a blood source, and thereby deprived of oxygen and nutrients for anextended period of time. Ischemic insult also occurs in certain clinicalconditions, such as sickle cell anemia and septic shock, which mayresult from hypotension or organ dysfunction. Depending on the durationof the insult, the ischemia can disturb cellular metabolism and iongradients, and ultimately cause irreversible cellular injury and death.

Arguably, heart attacks and stroke are the most widely recognizedexample of the damage resulting from ischemia. Myocardial ischemia is acondition wherein there is insufficient blood supply to the myocardium(the muscles of the heart) to meet its demand for oxygen. The ultimateresult of persistent myocardial ischemia is necrosis or death of aportion of cardiac muscle tissue, known as a myocardial infarct,commonly known as a heart attack. Insufficient blood supply to themyocardium is generally due to an obstruction or thrombus in an arterysupplying blood to the myocardium. Another cause can be atrialfibrillation, wherein the increased heart rate associated with atrialfibrillation increases the work, and hence the blood demand of themyocardium, while the atrial fibrillation at the same time reduces theblood supply.

Whereas stroke is defined as a sudden impairment of body functionscaused by a disruption in the supply of blood to the brain. Forinstance, a stroke occurs when blood supply to the brain is interruptedfor any reason, including hemorrhage, low blood pressure, clogging byatherosclerotic plaque, a blood clot, or any particle. Because of theblockage or rupture, part of the brain fails to get the supply of bloodand oxygen that it requires. Brain tissue that receives an inadequatesupply of blood is said to be ischemic. Deprived of oxygen andnutrients, nerve cells and other cell types within the brain begin tofail, creating an infarct (an area of cell death, or necrosis). As theneurons fail and die, the part of the body controlled by those neuronscan no longer function. The devastating effects of ischemia are oftenpermanent because brain tissue has very limited repair capabilities andlost neurons are typically not regenerated.

Cerebral ischemia may be incomplete (blood flow is reduced but notentirely cut off), complete (total loss of tissue perfusion), transientor permanent. If ischemia is incomplete and persists for no more thanten to fifteen minutes, neural death may not occur. More prolonged orcomplete ischemia results in infarction. Depending on the site andextent of the infarction, mild to severe neurological disability ordeath will follow.

To a modest extent, the brain is protected against cerebral ischemia bycompensatory mechanisms, including collateral circulation (overlappinglocal blood supplies), and arteriolar auto-regulation (local smoothmuscle control of blood flow in the smallest arterial channels). Ifcompensatory mechanisms operate efficiently, slightly diminishedcerebral blood flow produces neither tissue ischemia nor abnormal signsand symptoms. Usually, such mechanisms must act within minutes torestore blood flow if permanent infarction damage is to be avoided orreduced. Arteriolar auto-regulation works by shunting blood fromnoncritical regions to infarct zones.

Even in the face of systemic hypotension, auto-regulation may besufficient to adjust the circulation and thereby preserve the vitalityand function of brain or heart tissue. Alternatively, ischemia may besufficiently prolonged and compensatory mechanisms sufficientlyinadequate that a catastrophic stroke or heart attack results.

Ischemia is also associated with various clinical conditions, such asseptic shock. Septic shock as a result of hypotension and organdysfunction in response to infectious sepsis is a major cause of death.The manifestations of sepsis include those related to the systemicresponse to infection (tachycardia, tachypnea alterations in temperatureand leukocytosis) and those related to organ-system dysfunction(cardiovascular, respiratory, renal, hepatic and hematologicabnormalities). Furthermore, the lipopolysaccharide (LPS) ofgram-negative bacteria is considered to be the most important exogenousmediator of acute inflammatory response to septic shock. The LPS orendotoxin released from the outer membrane of gram-negative bacteriaresults in the release of cytokines and other cellular mediators,including tumor necrosis factor alpha (TNF alpha), interleukin-1 (Il-1),interleukin-6 (Il-6) and thromboxane A2. Extreme levels of thesemediators are known to trigger many pathological events, includingfever, shock, and intravascular coagulation, leading to ischemia andorgan failure.

II. Hemoglobin

Hemoglobin is a tetrameric protein which delivers oxygen via anallosteric mechanism. Oxygen binds to the four hemes of the hemoglobinmolecule. Each heme contains porphyrin and iron in the ferrous state.The ferrous iron-oxygen bond is readily reversible. Binding of the firstoxygen to a heme releases much greater energy than binding of the secondoxygen molecule, binding of the third oxygen releases even less energy,and binding of the fourth oxygen releases the least energy.

In blood, hemoglobin is in equilibrium between two allostericstructures. In the “T” (for tense) state, hemoglobin is deoxygenated. Inthe “R” (for relaxed) state, hemoglobin is oxygenated. An oxygenequilibrium curve can be scanned to observe the affinity and degree ofcooperativity (allosteric action) of hemoglobin. In the scan, the Y-axisplots the percent of hemoglobin oxygenation and the X-axis plots thepartial pressure of oxygen in millimeters of mercury (mm Hg). If ahorizontal line is drawn from the 50% oxygen saturation point to thescanned curve and a vertical line is drawn from the intersection pointof the horizontal line with the curve to the partial pressure X-axis, avalue commonly known as the P₅₀ is determined (i.e., this is thepressure in mm Hg when the scanned hemoglobin sample is 50% saturatedwith oxygen). Under physiological conditions (i.e., 37° C., pH=7.4, andpartial carbon dioxide pressure of 40 mm Hg), the P₅₀ value for normaladult hemoglobin (HbA) is around 26.5 mm Hg. If a lower than normal P₅₀value is obtained for the hemoglobin being tested, the scanned curve isconsidered to be “left-shifted” and the presence of high oxygen-affinityhemoglobin is indicated. Conversely, if a higher than normal P₅₀ valueis obtained for the hemoglobin being tested, the scanned curve isconsidered to be “right-shifted”, indicating the presence of lowoxygen-affinity hemoglobin.

It has been proposed that influencing the allosteric equilibrium ofhemoglobin is a viable avenue of attack for treating diseases. Theconversion of hemoglobin to a high affinity state is generally regardedto be beneficial in resolving problems with (deoxy)hemoglobin-S (i.e.,sickle cell anemia). The conversion of hemoglobin to a low affinitystate is believed to have general utility in a variety of disease stateswhere tissues suffer from low oxygen tension, such as ischemia and radiosensitization of tumors. Several synthetic compounds have beenidentified which have utility in the allosteric regulation of hemoglobinand other proteins. For example, several new compounds and methods fortreating sickle cell anemia which involve the allosteric regulation ofhemoglobin are reported in U.S. Pat. No. 4,699,926 to Abraham et al.,U.S. Pat. No. 4,731,381 to Abraham et al., U.S. Pat. No. 4,731,473 toAbraham et al., U.S. Pat. No. 4,751,244 to Abraham et al., and U.S. Pat.No. 4,887,995 to Abraham et al. Furthermore, in both Perutz, “Mechanismsof Cooperativity and allosteric Regulation in Proteins”, QuarterlyReviews of Biophysics 22, 2 (1989), pp. 163–164, and Lalezari et al.,“LR16, a compound with potent effects on the oxygen affinity ofhemoglobin, on blood cholesterol, and on low density lipoprotein”, Proc.Natl. Acad. Sci., USA 85 (1988), pp. 6117–6121, compounds which areeffective allosteric hemoglobin modifiers are discussed. In addition,Perutz et al. has shown that a known antihyperlipoproteinemia drug,bezafibrate, is capable of lowering the affinity of hemoglobin foroxygen (See “Bezafibrate lowers oxygen affinity of hemoglobin”, Lancet1983, 881).

Human normal adult hemoglobin (“HbA”) is a tetrameric protein comprisingtwo alpha chains having 141 amino acid residues each and two beta chainshaving 146 amino acid residues each, and also bearing prosthetic groupsknown as hemes. The erythrocytes help maintain hemoglobin in itsreduced, functional form. The heme-iron atom is susceptible tooxidation, but may be reduced again by one of two systems within theerythrocyte, the cytochrome b5, and glutathione reduction systems.

Hemoglobin is able to alter its oxygen affinity, thereby increasing theefficiency of oxygen transport in the body due to its dependence on2,3-DPG, an allosteric regulator. 2,3-DPG is present within erythrocytesat a concentration that facilitates hemoglobin to release bound oxygento tissues. Naturally-occurring hemoglobin includes any hemoglobinidentical to hemoglobin naturally existing within a cell.Naturally-occurring hemoglobin is predominantly wild-type hemoglobin,but also includes naturally-occurring mutant hemoglobin. Wild-typehemoglobin is hemoglobin most commonly found within natural cells.Wild-type human hemoglobin includes hemoglobin A, the normal adult humanhemoglobin having two alpha- and two beta-globin chains. Mutanthemoglobin has an amino-acid sequence that differs from the amino-acidsequence of wild-type hemoglobin as a result of a mutation, such as asubstitution, addition or deletion of at least one amino acid. Adulthuman mutant hemoglobin has an amino-acid sequence that differs from theamino-acid sequence of hemoglobin A. Naturally-occurring mutanthemoglobin has an amino-acid sequence that has not been modified byhumans. The naturally-occurring hemoglobin of the present invention isnot limited by the methods by which it is produced. Such methodstypically include, for example, erythrocytolysis and purification,recombinant production, and protein synthesis.

It is known that hemoglobin specifically binds small polyanionicmolecules, especially 2,3-diphosphoglycerate (DPG) and adenosinetriphosphate (ATP), present in the mammalian red cell (Benesch andBenesch, Nature, 221, p. 618, 1969). This binding site is located at thecentre of the tetrameric structure of hemoglobin (Arnone, A., Nature,237, p. 146, 1972). The binding of these polyanionic molecules isimportant in regulating the oxygen-binding affinity of hemoglobin sinceit allosterically affects the conformation of hemoglobin leading to adecrease in oxygen affinity (Benesch and Benesch, Biochem. Biophys. Res.Comm., 26, p. 162, 1967). Conversely, the binding of oxygenallosterically reduces the affinity of hemoglobin for the polyanion.(Oxy) hemoglobin therefore binds DPG and ATP weakly. This is shown, forexample, by studies of spin-labeled ATP binding to oxy- anddeoxyhemoglobin as described by Ogata and McConnell (Ann. N.Y. Acad.Sc., 222, p. 56, 1973). In order to exploit the polyanion-bindingspecificity of hemoglobin, or indeed to perform any adjustment of itsoxygen-binding affinity by chemically modifying the polyanion bindingsite, it has been necessary in the prior art that hemoglobin bedeoxygenated. However, hemoglobin as it exists in solutions, or mixturesexposed to air, is in its oxy state, i.e., (oxy)hemoglobin. In fact itis difficult to maintain hemoglobin solutions in the deoxy state,(deoxy)hemoglobin, throughout a chromatographic procedure. Because ofthese difficulties, the technique of affinity chromatography has notbeen used in the prior art to purify hemoglobin.

Hemoglobin has also been administered as a pretreatment to patientsreceiving chemotherapeutic agents or radiation for the treatment oftumors (U.S. Pat. No. 5,428,007; WO 92/20368; WO 92/20369), forprophylaxis or treatment of systemic hypotension or septic shock inducedby internal nitric oxide production (U.S. Pat. No. 5,296,466), duringthe perioperative period or during surgery in a method for maintaining asteady-state hemoglobin concentration in a patient (WO 95/03068), and aspart of a perioperative hemodilution procedure used prior to surgery inan autologous blood use method (U.S. Pat. Nos. 5,344,393 and 5,451,205).When a patient suffers a trauma (i.e., a wound or injury) resulting, forexample, from surgery, an invasive medical procedure, or an accident,the trauma disturbs the patient's homeostasis. The patient's bodybiologically reacts to the trauma to restore homeostasis. This reactionis referred to herein as a naturally occurring stress response. If thebody's stress response is inadequate or if it occurs well after thetrauma is suffered, the patient is more prone to develop disorders.

III. Reduction of the Oxygen-affinity of Hemoglobin

The major function of erythrocytes consists in the transport ofmolecular oxygen from the lungs to the peripheral tissues. Theerythrocytes contain a high concentration of hemoglobin (30 pg percell=35.5 g/100 ml cells) which forms a reversible adduct with O₂. TheO₂-partial pressure in the lung is about.100 mm Hg, in the capillarysystem is about.70 mm Hg, against which O₂ must be dissociated from theoxygenated hemoglobin. Under physiological conditions, only about 25% ofthe oxygenated hemoglobin may be deoxygenated; about 75% is carried backto the lungs with the venous blood. Thus, the major fraction of thehemoglobin-O₂ adduct is not used for the O₂ transport.

Interactions of hemoglobin with allosteric effectors enable anadaptation to the physiological requirement of maximum O₂ release fromthe hemoglobin-O₂ adduct with simultaneous conservation of the highestpossible O₂ partial pressure in the capillary system.2,3-Diphosphoglycerate increases the half-saturation pressure ofstripped hemoglobin at pH 7.4 from P(O₂) (½)=9.3 mm Hg (37° C.), and 4.3mm Hg (25° C.) to P(O₂) (½)=23.7 mm Hg (37° C.), and 12.0 mm Hg (25°C.), respectively (Imai, K. and Yonetani, T. (1975), J. Biol. Chem. 250,1093–1098). A significantly stronger decrease of the O₂ affinity, i.e.,enhancement of the O₂ half-saturation pressure has been achieved forstripped hemoglobin by binding of inositol hexaphosphate (phytic acid;IHP) (Ruckpaul, K. et al. (1971) Biochim. Biophys. Acta 236, 211–221)isolated from vegetal tissues. Binding of IHP to hemoglobin increasesthe O₂ half-saturation pressure to P(O₂) (½)=96.4 mm Hg (37° C.), andP(O₂) (½)=48.4 mm Hg (25° C.), respectively. IHP, like2,3-diphosphoglycerate and other polyphosphates cannot penetrate theerythrocyte membrane.

Furthermore, the depletion of DPG and ATP in stored red cells leads to aprogressive increase of the oxygen affinity of hemoglobin containedtherein (Balcerzak, S. et al. (1972) Adv. Exp. Med. Biol. 28, 453–447).The O₂-binding isotherms are measured in the absence of CO₂ and atconstant pH (pH 7.4) in order to preclude influences of these allostericeffectors on the half-saturation pressure. The end point of theprogressive polyphosphate depletion is defined by P(O₂) (½)=4.2 mm Hg,which is the half-saturation pressure of totally phosphate-free(stripped) hemoglobin; the starting point, i.e., P(O₂) (½) of fresherythrocytes, depends on the composition of the suspending medium. Fromthese polyphosphate depletion curves a new functional parameter ofstored erythrocytes can be determined, the so-called half-life time ofintra-erythrocytic polyphosphate: 9 d (days) in isotonic 0.1 M bis-Trisbuffer pH 7.4; and 12 d (days) in acid-citrate-dextrose conservation(ACD) solution.

Several years ago, it was discovered that the antilipidemic drugclofibric acid lowered the oxygen affinity of hemoglobin solutions(Abraham et al., J. Med. Chem. 25, 1015 (1982), and Abraham et al.,Proc. Natl. Acad. Sci. USA 80, 324 (1983)). Bezafibrate, anotherantilipidemic drug, was later found to be much more effective inlowering the oxygen affinity of hemoglobin solutions and suspensions offresh, intact red cells (Perutz et al., Lancet, 881, Oct. 15, 1983).Subsequently, X-ray crystallographic studies have demonstrated thatclofibric acid and bezafibrate bind to the same sites in the centralwater cavity of deoxyhemoglobin, and that one bezafibrate molecule willspan the sites occupied by two clofibric acid molecules. Bezafibrate andclofibric acid act by stabilizing the deoxy structure of hemoglobin,shifting the allosteric equilibrium toward the low affinity deoxy form.Bezafibrate and clofibric acid do not bind in any specific manner toeither oxy- or carbonmonoxyhemoglobin.

In more recent investigations, a series of urea derivatives[2-[4-[[(arylamino)carbonyl]amino]phenoxy]-2-methylpropionic acids] wasdiscovered that has greater allosteric potency than bezafibrate atstabilizing the deoxy structure of hemoglobin and shifting theallosteric equilibrium toward the low oxygen affinity form (Lalezari,Proc. Natl. Acad. Sci. USA 85, 6117 (1988)).

Drugs which can allosterically modify hemoglobin toward a lower oxygenaffinity state hold potential for many clinical applications, such asfor the treatment of ischemia, shock, and polycythemia, and asradiosensitizing agents. Unfortunately, the effects of bezafibrate andthe urea derivatives discussed above have been found to be significantlyinhibited by serum albumin, the major protein in blood serum (Lalezariet al., Biochemistry, 29, 1515 (1990)). Therefore, the clinicalusefulness of these drugs is seriously undermined because in whole bloodand in the body, the drugs would be bound by serum albumin instead ofreaching the red cells, crossing the red cell membrane, and interactingwith hemoglobin protein molecule to produce the desired effect.

There has been considerable interest in medicine, the military healthservices, and the pharmaceutical industry in finding methods to increaseblood storage life; to discover radio sensitization agents; and todevelop new blood substitutes. In all these instances, the availabilityof either autologous blood or recombinant Hb solutions is of majorinterest, provided the oxygen affinity can be decreased to enhanceoxygen delivery to the tissues.

2,3-Diphosphoglycerate (2,3-DPG) is the normal physiological ligand forthe allosteric site on hemoglobin. However, phosphorylated inositols arefound in the erythrocytes of birds and reptiles. Specifically, inositolhexaphosphate (IHP), as known as phytic acid, displaces hemoglobin-bound2,3-DPG, binding to the allosteric site with one-thousand times greateraffinity. Unfortunately, IHP is unable to pass unassisted across theerythrocyte membrane.

IV. Enhanced Oxygen Delivery in Mammals

The therapy of oxygen deficiencies requires the knowledge of parameterswhich characterize both the O₂ transport capacity and the O₂ releasecapacity of human RBCs. The parameters of the O₂ transport capacity,i.e., Hb concentration, the number of RBCs, and hemocrit, are commonlyused in clinical diagnosis. However, the equally important parameters ofthe O₂ release capacity, i.e., O₂ half-saturation pressure of Hb andRBCs, and the amounts of high and low oxygen affinity hemoglobins inRBCs, are not routinely determined and were not given seriousconsideration until pioneering work by Gerosonde and Nicolau (Blut,1979, 39, 1–7).

In the 1980s, Nicolau et al. (J. Appl. Physiol. 58:1810–1817 (1985);“PHYTIC ACID: Chemistry and Applications”; Graf, E., Ed.; Pilatus Press,Minneapolis, Minn., USA; 1986; and Proc. Natl. Acad. Sci. USA 1987, 84,6894–6898) reported that the encapsulation in red blood cells (RBCs) ofIHP, via a technique of controlled lysis and resealing, results in asignificant decrease in the hemoglobin affinity for oxygen. Theprocedure yielded RBCs with unchanged life spans, normal ATP and K+levels, and normal rheological competence. Enhancement of the O₂-releasecapacity of these cells brought about significant physiological effectsin piglets: 1) reduced cardiac output, linearly dependent on the P₅₀value of the RBCs; 2) increased arteriovenous difference; and 3)improved tissue oxygenation. Long term experiments showed that inpiglets the high P₅₀ value of IHP-RBCs was maintained over the entirelife spans of the RBCs.

More recently, Nicolau et al. (TRANSFUSION 1995, 35, 478–486; and U.S.Pat. No. 5,612,207) reported the use of a large-volume, continuous-flowelectroporation system for the encapsulating IHP in human RBCs. Thesemodified RBCs possess P₅₀ values of approximately 50 torr, roughly twicethat of unmodified human RBCs. Additionally, 85% of the RBCs survivedthe electroporation process, displaying hematologic indices nearlyidentical to those of unmodified RBCs. Nicolau's electroporation systemprocesses one unit of blood every ninety minutes.

Although it is evident that methods of enhancing oxygen delivery totissues have potential medical applications, currently there are nomethods clinically available for increasing tissue delivery of oxygenbound to hemoglobin. Transient, e.g., 6 to 12 hour, elevations of oxygendeposition have been described in experimental animals using either DPGor molecules that are precursors of DPG. However, the natural regulationof DPG synthesis in vivo and its relatively short biological half-lifelimit the DPG concentration and the duration of increased tissue P(O₂),and thus limit its therapeutic usefulness.

Additionally, as reported in Genetic Engineering News, Vol. 12, No. 6,Apr. 15, 1992, several groups are attempting to engineer freeoxygen-carrying hemoglobin as a replacement for human blood.Recombinant, genetically modified human hemoglobin that does not breakdown in the body and that can readily release up to 30% of its boundoxygen is currently being tested by Somatogen, Inc., of Boulder Colo.While this product could be useful as a replacement for blood lost intraumatic injury or surgery, it would not be effective to increase PO₂levels in ischemic tissue, since its oxygen release capacity isequivalent to that of natural hemoglobin (27–30%). As are allrecombinant products, this synthetic hemoglobin is also likely to be acostly therapeutic.

Synthetic human hemoglobin has also been produced in neonatal pigs byinjection of human genes that control hemoglobin production. Thisproduct may be a less expensive product than the Somatogen synthetichemoglobin, but it does not solve problems with oxygen affinity andbreakdown of hemoglobin in the body.

V. Specific Clinical Applications of Enhanced Oxygen Delivery

There are numerous clinical conditions that would benefit fromtreatments that would increase tissue delivery of oxygen bound tohemoglobin. For example, the leading cause of death in the United Statestoday is cardiovascular disease. The acute symptoms and pathology ofmany cardiovascular diseases, including congestive heart failure,myocardial infarction, stroke, intermittent claudication, and sicklecell anemia, result from an insufficient supply of oxygen in fluids thatbathe the tissues. Likewise, the acute loss of blood followinghemorrhage, traumatic injury, or surgery results in decreased oxygensupply to vital organs. Without oxygen, tissues at sites distal to theheart, and even the heart itself, cannot produce enough energy tosustain their normal functions. The result of oxygen deprivation istissue death and organ failure.

Although the attention of the American public has long been focused onthe preventive measures required to alleviate heart disease, such asexercise, appropriate dietary habits, and moderation in alcoholconsumption, deaths continue to occur at an alarming rate. Since deathresults from oxygen deprivation, which in turn results in tissuedestruction and/or organ dysfunction, one approach to alleviate thelife-threatening consequences of cardiovascular disease is to increaseoxygenation of tissues during acute stress. The same approach is alsoappropriate for persons suffering from blood loss or chronic hypoxicdisorders, such as congestive heart failure.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to inositol hexaphosphate(IHP) derivatives comprising an internal pyrophosphate moiety. Anotheraspect of the present invention relates to compositions, consistingessentially of aliphatic ammonium cations or of metal cations, such assodium cations, and IHP derivatives comprising an internal pyrophosphatemoiety. The present invention also relates to methods for modulating theoxygen affinity of hemoglobins comprising the use as allostericeffectors of hemoglobin of the aforementioned IHP deriviatives and thecompositions comprising them.

An aliphatic ammonium cation is substituted with one or more aliphaticgroups, which can be the same or different. In certain embodiments, thealiphatic ammonium cation is a primary ammonium cation represented bythe general formula RN⁺H₃, wherein R is an aliphatic group, preferablyan alkyl, preferably a lower alkyl, i.e., a C₁–C₈ alkyl, and morepreferably a C₃–C₁₀ cycloalkyl. In certain preferred embodiments, theammonium cation is derived from cyclic amines.

In certain embodiments, the present invention relates to compounds, andcompositions thereof, that deliver IHP into erythrocytes in vivo or exvivo, for lowering the oxygen affinity of hemoglobin in red blood cellsuspensions and whole blood. It is an object of this invention toprovide methods for delivering IHP into erythrocytes in whole blood,utilizing compounds or compositions thereof that do not lose theireffectiveness in the presence of whole blood.

In certain embodiments, the present invention relates to a method oftreating a subject suffering from one or more diseases where an increasein oxygen delivery of hemoglobin would be beneficial, comprising thesteps of treating red blood cells or whole blood ex vivo with one ormore compounds or compositions of the present invention, followed bysuitably purifying said red blood cells or whole blood, andadministering the prepared red blood cells or whole blood to saidsubject. By ‘suitably purifying’ it is meant a method of washing andseparating, for example by centrifugation, the red blood cell-allostericeffector or whole blood-allosteric effector suspension, and discardingthe supernatant until no non-encapsulated allosteric effector can bedetected. An exemplary method is presented in detail by Nicolau et al.in U.S. Pat. No. 5,612,207, which is incorporated by reference herein.

Ligands for the allosteric site of hemoglobin interact with thehemoglobin molecule and impact its ability to bind oxygen. Thisinvention is particularly concerned with the delivery of IHP derivativescomprising an internal pyrophosphate moiety, thereby causing oxygen tobe bound relatively less tightly to hemoglobin, such that oxygen isoff-loaded from the hemoglobin molecule more easily.

The process of allosterically modifying hemoglobin towards a loweroxygen affinity state in whole blood may be used in a wide variety ofapplications, including treatments for ischemia, heart disease, woundhealing, radiation therapy of cancer, and adult respiratory distresssyndrome (ARDS). Furthermore, a decrease in the oxygen affinity ofhemoglobin in whole blood will extend its useful shelf-life vis-à-vistransfusions, and/or restore the oxygen carrying capacity of aged blood.

Another condition which could benefit from an increase in the deliveryof oxygen to the tissues is anemia. A significant portion of hospitalpatients experience anemia or a low “crit” caused by an insufficientquantity of red blood cells or hemoglobin in their blood. This leads toinadequate oxygenation of their tissues and subsequent complications.Typically, a physician can temporarily correct this condition bytransfusing the patient with units of packed red blood cells.

Enhanced blood oxygenation may also reduce the number of heterologoustransfusions and allow use of autologous transfusions in more cases. Thecurrent method for treatment of anemia or replacement of blood loss istransfusion of whole human blood. It is estimated that three to fourmillion patients receive transfusions in the U.S. each year for surgicalor medical needs. In situations where there is more time it isadvantageous to avoid the use of donor or heterologous blood, insteadusing autologous blood. However, often the amount of blood which can bedrawn and stored prior to surgery limits the use of autologous blood.Typically, a surgical patient does not have enough time to donate asufficient quantity of blood prior to surgery. A surgeon would like tohave several units of blood available. As each unit requires a period ofseveral weeks between donations, and because a unit can not be drawnless than two weeks prior to surgery, it is often impossible tosequester an adequate supply of blood. By processing autologous bloodwith an IHP derivative comprising an internal pyrophosphate moiety, lessblood is required and it becomes possible to avoid the transfusion ofheterologous blood.

Because IHP-treated RBCs may release up to 2–3 times as much oxygen asuntreated red cells, in many cases, a physician will need to transfusefewer units of IHP-treaded red cells. This exposes the patient to lessheterologous blood, decreases the extent of exposure to diseases fromblood donors and minimizes immune function disturbances secondary totransfusions. The ability to infuse more efficient red blood cells isalso advantageous when the patients blood volume is excessive. In moresevere cases, where oxygen transport is failing, the ability to improverapidly a patient's tissue oxygenation may be life saving.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the ³¹P NMR spectrum of IHP-cholesteryloxy carbonyl heptaN,N-dimethylcyclohexylammonium salt and the ³¹P NMR spectrum ofIHP-tripyrophosphate (“ITPP”).

FIG. 2 depicts the crude and purified ³¹P NMR spectra ofIHP-cholesteryloxy carbonyl in various deuterated solvents.

FIG. 3 depicts the ³¹P NMR spectrum of purified IHP-cholesteryloxycarbonyl; the ³¹P NMR spectrum of highly purified IHP-cholesteryloxycarbonyl; the ³¹P NMR spectrum of IHP-monopyrophosphate (“IMPP”); andthe ³¹P NMR spectrum of ITPP.

FIG. 4 depicts the ³¹P NMR spectra of IHP-benzoate in different solventsand the ³¹P NMR spectrum of IHP-benzoate after heating.

FIG. 5 depicts the ³¹P NMR spectrum of crude IHP-benzoate; the ³¹P NMRspectrum of purified IHP-benzoate; and the ³¹P NMR spectrum ofIHP-benzoate at pH 6.9.

FIG. 6 depicts the ³¹P NMR spectrum of the crude IHP-hexanoylderivative; the ³¹P NMR spectrum of the purified IHP-hexanoylderivative; the ³¹P NMR spectrum of the IHP-hexanoyl derivative at pH7.3; and the ³¹P NMR spectrum of the IHP-hexanoyl derivative afterheating.

FIG. 7 depicts the ³¹P NMR spectra of kf56, kf53, kf31, kf31A,respectively.

FIG. 8 depicts 2a): mixed ³¹P NMR of all compounds 5 (kf31, kf53, kf56);2b): 2D ³¹P NMR COSY experiment of compound kf53.

FIG. 9 depicts ITPP uptake in 1-octanol by cyclooctylammonium ions.

FIG. 10 depicts the general scheme for the synthesis of IHP derivatives.

FIG. 11 depicts means of 12 P₅₀O-values and standard deviation areshown. On day 4 the kf111-solution was replaced by water. P₅₀ valueswere measured over 12 days. Four mice received IHP in water, at the sameconcentration as kf111. On day 4 IHP was replaced by water. Three micereceived only water during the 12 days.

FIG. 12 depicts P₅₀ shifts of 4 single mice (and standard deviation areshown). Mouse 1, Mouse 12, IHP control mouse, water control mouse.

FIG. 13 depicts the relation of P₅₀ shift [%] to erythrocytes (valuesare taken from Table 1). Based upon the preliminary data reported thatan inverse relationship exists between the number of RBC and shift oftheir P₅₀ value. The basal value of the RBC count is restored, once P₅₀becomes 0%, 12 days after ingestion of kf111.

FIG. 14 depicts the P₅₀ shifts (means of 4 measurements) in blood froman injected, and a non injected piglet and standard deviations areshown. Value obtained on day 0=measurement 2.5 hrs after injection.

FIG. 15 depicts the dosis curve for 3 piglets injected via iv with 0.3,0.5, 1*, 1.3 and 1.5 g kf111 per kg body weight. Means of 4 single P₅₀values per blood sample and standard deviation are given. (*2 pigletsinjected).

DETAILED DESCRIPTION OF THE INVENTION

I. Overview

The process of allosterically modifying hemoglobin towards a low oxygenaffinity state in whole blood could be used in a wide variety ofapplications including in treatments for ischemia, heart disease,complications associated with angioplasty, wound healing, radiationtherapy of cancer, adult respiratory distress syndrome (ARDS), etc., inextending the shelf-life of blood or restoring the oxygen carryingcapacity of out-dated blood, and as sensitizers for x-ray irradiation incancer therapy, as well as in many other applications.

This invention is related to the use of allosteric hemoglobin modifiercompounds in red blood cell suspensions, e.g., in whole blood. Serumalbumin, which is the most abundant protein in blood plasma, has beenidentified as inhibiting the allosteric effects of clofibric acid,bezafibrate, and L3,5/L3,4,5. The precise nature of this inhibition isnot fully understood, but appears to be related to these compoundsbinding to the serum albumin. In contrast, the subject compounds havebeen found to be relatively unaffected by the presence of serum albumin.Ligands for the allosteric site of hemoglobin that are not adverselyeffected by serum albumin represent particularly good candidates fordrug applications, since the performance of the drug will not befrustrated by the presence of serum albumin present in a patient'sblood.

This invention relates to the incorporation of a wide variety oftherapeutically useful substances into mammalian red blood cells (RBCs),which could not previously be accomplished without unacceptable lossesof RBC contents and/or integrity. In particular, the compounds andmethods of the present invention make possible the introduction orincorporation into RBCs of anionic agents, such as DNA, RNA,chemotherapeutic agents, and antibiotic agents. These and otherwater-soluble substances may be used for a desired slow continuousdelivery or targeted delivery when the treated and purified RBC carrieris later injected in vivo. The particular anion or polyanion to beselected can be based on whether an allosteric effector of hemoglobinwould be desirable for a particular treatment.

The present invention provides a novel method for increasing theoxygen-carrying capacity of erythrocytes. In accordance with the methodof the present invention, the IHP combines with hemoglobin in a stableway, and shifts its oxygen releasing capacity. Erythrocytes withIHP-hemoglobin can release more oxygen per molecule than hemoglobinalone, and thus more oxygen is available to diffuse into tissues foreach unit of blood that circulates. IHP is preferably added to red bloodcells in vitro or ex vivo, as it appears that it is toxic to animalsunder certain circumstances.

Another advantage of IHP-treated red blood cells is that they show theBohr effect in circulation and when stored. Normal red blood cells thathave been stored do not regain their maximum oxygen carrying capacity incirculation for approximately 24 hours. This is because the DPG presentin normal red blood cells is degraded by native enzymes, e.g.,phosphatases, during storage and must be replaced by the body aftertransfusion. In contrast, red blood cells treated according to thepresent invention retain their maximum oxygen carrying capacity duringstorage and therefore can deliver oxygen to the tissues in response todemand immediately after transfusion into a human or animal becausethere are no native enzymes in erythrocytes which degrade IHP.

IHP-treated RBCs may be used in the treatment of acute and chronicconditions, including, but not limited to, hospitalized patients,cardiovascular operations, chronic anemia, anemia following majorsurgery, coronary infarction and associated problems, chronic pulmonarydisease, cardiovascular patients, autologous transfusions, as anenhancement to packed red blood cells transfusion (hemorrhage, traumaticinjury, or surgery) congestive heart failure, myocardial infarction(heart attack), stroke, peripheral vascular disease, intermittentclaudication, circulatory shock, hemorrhagic shock, anemia and chronichypoxia, respiratory alkalemia, metabolic alkalosis, sickle cell anemia,reduced lung capacity caused by pneumonia, surgery, complicationsassociated with angioplasty, pneumonia, trauma, chest puncture,gangrene, anaerobic infections, blood vessel diseases such as diabetes,substitute or complement to treatment with hyperbaric pressure chambers,intra-operative red cell salvage, cardiac inadequacy, anoxia-secondaryto chronic indication, organ transplant, carbon monoxide, nitric oxide,and cyanide poisoning.

This invention is related to a method of treating a subject for any oneor more of the above diseases comprising the steps of treating red bloodcells or whole blood ex vivo with one or more compounds or compositionsof the present invention, followed by suitably purifying said red bloodcells or whole blood, and administering the thus prepared red bloodcells or whole blood to said subject. By ‘suitably purifying’ it ismeant a method of washing and separating the red blood cell- or wholeblood-allosteric effector suspension and discarding the supernatantuntil no non-encapsulated allosteric effector can be detected, e.g., asdevised by Nicolau et al. in U.S. Pat. No. 5,612,207. Alternatively, acompound comprised of an allosteric effector can be administereddirectly to a subject if the compound does not have toxic effects in thesubject, or at least its beneficial effects predominate over itstoxicity in a subject. Toxicity of a compound in a subject can bedetermined according to methods known in the art.

Treating a human or animal for any one or more of the above diseasestates is done by transfusing into the human or animal betweenapproximately 0.1 and 6 units (1 unit=500 mL) of IHP-treated blood thathas been prepared according to the present invention. In certain cases,blood exchange with IHP-treated blood may be possible. The volume ofIHP-treated red blood cells that is administered to the human or animalwill depend upon the value of P₅₀ for the IHP-treated RBCs. It is to beunderstood that the volume of IHP-treated red blood cells that isadministered to the patient can vary and still be effective. IHP-treatedRBCs are similar to normal red blood cells in every respect except thattheir P₅₀ value is shifted towards higher partial pressures of O₂.Erythrocytes release oxygen only in response to demand by organs andtissue. Therefore, the compounds, compositions thereof, and methods ofthe present invention will only restore a normal level of oxygenation tohealthy tissue, avoiding the cellular damage that is associated with anover-abundance of oxygen.

Because the compounds, compositions, and methods of the presentinvention are capable of allosterically modifying hemoglobin to favorthe low oxygen affinity “T” state (i.e., right shifting the equilibriumcurve), RBC's or whole blood treated with the compounds of the presentinvention and subsequently purified will be useful in treating a varietyof disease states in mammals, including humans, wherein tissues sufferfrom low oxygen tension, such as cancer and ischemia. Furthermore, asdisclosed by Hirst et al. (Radiat. Res., 112, (1987), pp. 164),decreasing the oxygen affinity of hemoglobin in circulating blood hasbeen shown to be beneficial in the radiotherapy of tumors. RBC's orwhole blood treated with the compounds of the present invention andsubsequently purified may be administered to patients in whom theaffinity of hemoglobin for oxygen is abnormally high. For example,certain hemoglobinopathies, certain respiratory distress syndromes,e.g., respiratory distress syndromes in new born infants aggravated byhigh fetal hemoglobin levels, and conditions in which the availabilityof hemoglobin/oxygen to the tissues is decreased (e.g., in ischemicconditions such as peripheral vascular disease, coronary occlusion,cerebral vascular accidents, or tissue transplant). The compounds andcompositions may also be used to inhibit platelet aggregation,antithrombotic purposes, and wound healing.

Additionally, the compounds and compositions of the present inventioncan be added to whole blood or packed cells preferably at the time ofstorage or at the time of transfusion in order to facilitate thedissociation of oxygen from hemoglobin and improve the oxygen deliveringcapability of the blood. When blood is stored, the hemoglobin in theblood tends to increase its affinity for oxygen by losing2,3-diphosphoglycerides. As described above, the compounds andcompositions of this invention are capable of reversing and/orpreventing the functional abnormality of hemoglobin observed when wholeblood or packed cells are stored. The compounds and compositions may beadded to whole blood or red blood cell fractions in a closed systemusing an appropriate reservoir in which the compound or composition isplaced prior to storage or which is present in the anticoagulatingsolution in the blood collecting bag.

Administration to a patient can be achieved by intravenous orintraperitoneal injection where the dose of treated red blood cells orwhole blood and the dosing regiment is varied according to individual'ssensitivity and the type of disease state being treated. Solid tumorsare oxygen deficient masses. The compounds, compositions and methods ofthis invention may be exploited to cause more oxygen to be delivered totumors, increasing radical formation and thereby increasing tumorkilling during radiation. In this context, such IHP-treated blood willonly be used in conjunction with radiotherapy.

The compounds, compositions and methods of this invention may beexploited to cause more oxygen to be delivered at low blood flow and lowtemperatures, providing the ability to decrease or prevent the cellulardamage, e.g., myocardial or neuronal, typically associated with theseconditions.

The compounds, compositions and methods of this invention may beexploited to decrease the number of red blood cells required fortreating hemorrhagic shock by increasing the efficiency with which theydeliver oxygen.

Damaged tissues heal faster when there is better blood flow andincreased oxygen tension. Therefore, the compounds, compositions andmethods of this invention may be exploited to speed wound healing.Furthermore, by increasing oxygen delivery to wounded tissue, thecompounds, compositions and methods of this invention may play a role inthe destruction of infection causing bacteria at a wound.

The compounds, compositions and methods of this invention may beeffective in enhancing the delivery oxygen to the brain, especiallybefore complete occlusion and reperfusion injuries occur due to freeradical formation. Furthermore, the compounds, compositions and methodsof this invention of this invention should reduce the expansion ofarterioles under both hypoxic and hypotensive conditions.

The compounds, compositions and methods of this invention of thisinvention should be capable of increasing oxygen delivery to blockedarteries and surrounding muscles and tissues, thereby relieving thedistress of angina attacks.

Acute respiratory disease syndrome (ARDS) is characterized byinterstitial and/or alveolar edema and hemorrhage as well asperivascular lung edema associated with the hyaline membrane,proliferation of collagen fibers, and swollen epithelium with increasedpinocytosis. The enhanced oxygen delivering capacity provided to RBCs bythe compounds, compositions and methods of this invention could be usedin the treatment and prevention of ARDS by militating against lower thannormal oxygen delivery to the lungs.

There are several aspects of cardiac bypass surgery that make attractivethe use of compounds or compositions or methods of the presentinvention. First, the compounds and compositions of the presentinvention may act as neuroprotective agents. After cardiac bypasssurgery, up to 50–70% of patients show some signs of cerebral ischemiabased on tests of cognitive function. Up to 5% of these patients haveevidence of stroke. Second, cardioplegia is the process of stopping theheart and protecting the heart from ischemia during heart surgery.Cardioplegia is performed by perfusing the coronary vessels withsolutions of potassium chloride and bathing the heart in ice water.However, blood cardioplegia is also used. This is where potassiumchloride is dissolved in blood instead of salt water. During surgery theheart is deprived of oxygen and the cold temperature helps slow downmetabolism. Periodically during this process, the heart is perfused withthe cardioplegia solution to wash out metabolites and reactive species.Cooling the blood increases the oxygen affinity of its hemoglobin, thusmaking oxygen unloading less efficient. However, treatment of bloodcardioplegia with RBC's or whole blood previously treated with compoundsor compositions of the present invention and subsequently purified willcounteract the effects of cold on oxygen affinity and make oxygenrelease to the ischemic myocardium more efficient, possibly improvingcardiac function after the heart begins to beat again. Third, duringbypass surgery the patient's blood is diluted for the process of pumpprime. This hemodilution is essentially acute anemia. Because thecompounds and compositions of the present invention make oxygentransport more efficient, their use during hemodilution (whether inbypass surgery or other surgeries, such as orthopedic or vascular) wouldenhance oxygenation of the tissues in an otherwise compromisedcondition. Additionally, the compounds and methods of the presentinvention will also find use in patients undergoing angioplasty, who mayexperience acute ischemic insult, e.g., due to the dye(s) used in thisprocedure.

Additionally, microvascular insufficiency has been proposed by a numberof investigators as a possible cause of diabetic neuropathy. Theinterest in microvascular derangement in diabetic neuropathic patientshas arisen from studies suggesting that absolute or relative ischemiamay exist in the nerves of diabetic subjects due to altered function ofthe endo- and/or epineurial blood vessels. Histopathologic studies haveshown the presence of different degrees of endoneurial and epineurialmicrovasculopathy, mainly thickening of blood vessel wall or occlusion.A number of functional disturbances have also been demonstrated in themicrovasculature of the nerves of diabetic subjects. Studies havedemonstrated decreased neural blood flow, increased vascular resistance,decreased pO₂ and altered vascular permeability characteristics such asa loss of the anionic charge barrier and decreased charge selectivity.Abnormalities of cutaneous blood flow correlate with neuropathy,suggesting that there is a clinical counterpart to the microvascularinsufficiency that may prove to be a simple non-invasive test of nervefiber dysfunction. Accordingly, patients suffering from diabeticneuropathies and/or other neurodegenerative disorders will likelybenefit from treatment based on the compounds and methods of the presentinvention.

Red blood cells or whole blood previously treated with the compounds ofthe present invention and subsequently suitably purified may be used toenhance oxygen delivery in any organism, e.g., fish, that uses ahemoglobin with an allosteric binding site.

II. Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. As used throughout thisspecification and the claims, the following terms have the followingmeanings:

The term “hemoglobin” includes all naturally- andnon-naturally-occurring hemoglobin.

The term “hemoglobin preparation” includes hemoglobin in aphysiologically compatible carrier or lyophilized hemoglobinreconstituted with a physiologically compatible carrier, but does notinclude whole blood, red blood cells or packed red blood cells.

The term “toxic” refers to a property where the deleterious effects aregreater than the beneficial effects.

The term “nontoxic” refers to a property where the beneficial effectsare greater than the deleterious effects.

The term “whole blood” refers to blood containing all its naturalconstituents, components, or elements or a substantial amount of thenatural constituents, components, or elements. For example, it isenvisioned that some components may be removed by the purificationprocess before administering the blood to a subject.

“Purified”, “purification process”, and “purify” all refer to a state orprocess of removing one or more compounds of the present invention fromthe red blood cells or whole blood such that when administered to asubject the red blood cells or whole blood is nontoxic.

“Non-naturally-occurring hemoglobin” includes synthetic hemoglobinhaving an amino-acid sequence different from the amino-acid sequence ofhemoglobin naturally existing within a cell, and chemically-modifiedhemoglobin. Such non-naturally-occurring mutant hemoglobin is notlimited by its method of preparation, but is typically produced usingone or more of several techniques known in the art, including, forexample, recombinant DNA technology, transgenic DNA technology, proteinsynthesis, and other mutation-inducing methods.

“Chemically-modified hemoglobin” is a natural or non-natural hemoglobinmolecule which is bonded to another chemical moiety. For example, ahemoglobin molecule can be bonded to pyridoxal-5′-phosphate, or otheroxygen-affinity-modifying moiety to change the oxygen-bindingcharacteristics of the hemoglobin molecule, to crosslinking agents toform crosslinked or polymerized hemoglobin, or to conjugating agents toform conjugated hemoglobin.

“Oxygen affinity” means the strength of binding of oxygen to ahemoglobin molecule. High oxygen affinity means hemoglobin does notreadily release its bound oxygen molecules.

The P₅₀ is a measure of oxygen affinity.

“Cooperativity” refers to the sigmoidal oxygen-binding curve ofhemoglobin, i.e., the binding of the first oxygen to one subunit withinthe tetrameric hemoglobin molecule enhances the binding of oxygenmolecules to other unligated subunits. It is conveniently measured bythe Hill coefficient (n[max]). For Hb A, n[max]=3.0.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure. “Ischemia” means a temporary or prolonged lack or reduction ofoxygen supply to an organ or skeletal tissue. Ischemia can be inducedwhen an organ is transplanted, or by conditions such as septic shock andsickle cell anemia.

“Skeletal tissue” means the substance of an organic body of a skeletalorganism consisting of cells and intercellular material, including butnot limited to epithelium, the connective tissues (including blood, boneand cartilage), muscle tissue, and nerve tissue.

“Ischemic insult” means damage to an organ or skeletal tissue caused byischemia.

“Subject” means any living organism, including humans, and mammals.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrastemal injection and infusion.

As used herein, the term “surgery” refers to the treatment of diseases,injuries, and deformities by manual or operative methods. Commonsurgical procedures include, but are not limited to, abdominal, aural,bench, cardiac, cineplastic, conservative, cosmetic, cytoreductive,dental, dentofacial, general, major, minor, Moh's, open heart, organtransplantation, orthopedic, plastic, psychiatric, radical,reconstructive, sonic, stereotactic, structural, thoracic, andveterinary surgery. The method of the present invention is suitable forpatients that are to undergo any type of surgery dealing with anyportion of the body, including but not limited to those described above,as well as any type of any general, major, minor, or minimal invasivesurgery.

“Minimally invasive surgery” involves puncture or incision of the skin,or insertion of an instrument or foreign material into the body.Non-limiting examples of minimal invasive surgery include arterial orvenous catheterization, transurethral resection, endoscopy (e.g.,laparoscopy, bronchoscopy, uroscopy, pharyngoscopy, cystoscopy,hysteroscopy, gastroscopy, coloscopy, colposcopy, celioscopy,sigmoidoscopy, and orthoscopy), and angioplasty (e.g., balloonangioplasty, laser angioplasty, and percutaneous transluminalangioplasty).

The term “ED₅₀” means the dose of a drug that produces 50% of itsmaximum response or effect. Alternatively, the dose that produces apre-determined response in 50% of test subjects or preparations.

The term “LD₅₀” means the dose of a drug that is lethal in 50% of testsubjects.

The term “therapeutic index” refers to the therapeutic index of a drugdefined as LD₅₀/ED₅₀.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

The term “structure-activity relationship (SAR)” refers to the way inwhich altering the molecular structure of drugs alters their interactionwith a receptor, enzyme, etc.

The term “pyrophosphate” refers to the general formula below:

wherein R is selected independently for each ocurrence from the groupconsisting of H, cations and hydrocarbon groups.

The terms “internal pyrophosphate moiety”, “internal pyrophosphatering”, and “cyclic pyrophosphate” refer to the structure feature below:

wherein R is selected independently for each ocurrence from the groupconsisting of H, cations, alkyl, alkenyl, alkynyl, aralkyl, aryl, andacyl groups.

The term “IHP-monopyrophosphate” (abbreviated as “IMPP”) refers toinositol hexaphosphate where two orthopyrophosphates were condensed toone internal pyrophosphate ring.

The term “IHP-tripyrophosphate” or “inositol tripyrophosphate” (bothabbreviated as “ITPP”) refers to inositol hexaphosphate with threeinternal pyrophosphate rings.

The term “2,3-diphosph-D-glyceric acid” (DPG) refers to the compoundbelow:

The term “2,3-cyclopyrophosphoglycerate” (CPPG) refers to the compoundbelow:

The term “ammonium cation” refers to the structure below:

wherein R represents independently for each occurrence H or asubstituted or unsubstituted aliphatic group. An “aliphatic ammoniumcation” refers to the above structure when at least one R is analiphatic group. A “quaternary ammonium cation” refers to the abovestructure when all four occurrences of R independently representaliphatic groups. R can be the same for two or more occurrences, ordifferent for all four.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are boron, nitrogen,oxygen, phosphorus, sulfur and selenium.

The term “electron-withdrawing group” is recognized in the art, anddenotes the tendency of a substituent to attract valence electrons fromneighboring atoms, i.e., the substituent is electronegative with respectto neighboring atoms. A quantification of the level ofelectron-withdrawing capability is given by the Hammett sigma (σ)constant. This well known constant is described in many references, forinstance, J. March, Advanced Organic Chemistry, McGraw Hill BookCompany, New York, (1977 edition) pp. 251–259. The Hammett constantvalues are generally negative for electron donating groups (σ[P]=−0.66for NH₂) and positive for electron withdrawing groups (σ[P]=0.78 for anitro group), σ[P] indicating para substitution. Exemplaryelectron-withdrawing groups include nitro, acyl, formyl, sulfonyl,trifluoromethyl, cyano, chloride, and the like. Exemplaryelectron-donating groups include amino, methoxy, and the like.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁–C₃₀ for straight chain, C₃–C₃₀ for branchedchain), and more preferably 20 or fewer. Likewise, preferred cycloalkylshave from 3–10 carbon atoms in their ring structure, and more preferablyhave 5, 6 or 7 carbons in the ring structure.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Preferred alkyl groups are lower alkyls. Inpreferred embodiments, a substituent designated herein as alkyl is alower alkyl.

The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls.

The terms ortho, meta and para apply to 1,2-, 1,3- and 1,4-disubstitutedbenzenes, respectively. For example, the names 1,2-dimethylbenzene andortho-dimethylbenzene are synonymous.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety, —CF₃, —CN, or the like.

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀ and R′₁₀ each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R₈, or R₉ and R₁₀ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R₈ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In preferred embodiments, only one of R₉ or R₁₀can be a carbonyl, e.g., R₉, R₁₀ and the nitrogen together do not forman imide. In even more preferred embodiments, R₉ and R₁₀ (and optionallyR′₁₀) each independently represent a hydrogen, an alkyl, an alkenyl, or—(CH₂)_(m)—R₈. Thus, the term “alkylamine” as used herein means an aminegroup, as defined above, having a substituted or unsubstituted alkylattached thereto, i.e., at least one of R₉ and R₁₀ is an alkyl group.

The term “acylamino” is art-recognized and refers to a moiety that canbe represented by the general formula:

wherein R₉ is as defined above, and R′₁₁ represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉, R₁₀ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

The term “carbonyl” is art recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₈ or apharmaceutically acceptable salt, R′₁₁ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above. WhereX is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R₁₁ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₁₁ is a hydrogen, the formula represents a “carboxylic acid”. Where Xis an oxygen, and R′₁₁ is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiolcarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thiolester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiolcarboxylic acid.” Where X is a sulfur and R₁₁′ ishydrogen, the formula represents a “thiolformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R₈,where m and R₈ are described above.

The term “sulfonate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is as defined above.

The term “sulfonamido” is art recognized and includes a moiety that canbe represented by the general formula:

in which R₉ and R′₁₁ are as defined above.

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₉ and R₁₀ are as defined above.

The term “sulfonyl”, as used herein, refers to a moiety that can berepresented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

The term “sulfoxido” as used herein, refers to a moiety that can berepresented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e.g. alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991).

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof, wherein one or more simple variations ofsubstituents are made which do not adversely affect the efficacy of thecompound. In general, the compounds of the present invention may beprepared by the methods illustrated in the general reaction schemes as,for example, described below, or by modifications thereof, using readilyavailable starting materials, reagents and conventional synthesisprocedures. In these reactions, it is also possible to make use ofvariants which are in themselves known, but are not mentioned here.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986–87, inside cover.

III. Compounds of the Invention.

Several years ago, it was discovered that the antilipidemic drugclofibric acid lowered the oxygen affinity of hemoglobin solutions(Abraham et al., J. Med. Chem. 25, 1015 (1982), and Abraham et al.,Proc. Natl. Acad. Sci. USA 80, 324 (1983)). Bezafibrate, anotherantilipidemic drug, was later found to be much more effective inlowering the oxygen affinity of hemoglobin solutions and suspensions offresh, intact red cells (Perutz et al., Lancet, 881, Oct. 15, 1983).Subsequently, X-ray crystallographic studies have demonstrated thatclofibric acid and bezafibrate bind to the same sites in the centralwater cavity of deoxyhemoglobin, and that one bezafibrate molecule willspan the sites occupied by two clofibric acid molecules. Bezafibrate andclofibric acid act by stabilizing the deoxy structure of hemoglobin,shifting the allosteric equilibrium toward the low affinity deoxy form.Bezafibrate and clofibric acid do not bind in any specific manner toeither oxy- or carbonmonoxyhemoglobin.

In later investigations, a series of urea derivatives[2-[4-[[(arylamino)carbonyl]-amino]phenoxy]-2-methylpropionic acids] wasdiscovered that has greater allosteric potency than bezafibrate atstabilizing the deoxy structure of hemoglobin and shifting theallosteric equilibrium toward the low oxygen affinity form (Lalezari,Proc. Natl. Acad. Sci. USA 85, 6117 (1988)).

It has been determined that certain allosteric hemoglobin modifiercompounds are hydrophobic molecules that can be bound to the body'sneutral fat deposits and lipophilic receptors sites, thus lowering theirpotency due to a decreased concentration in RBCs. Administration of ahydrophobic compound, such as a mixture of anesthetic molecules, willsaturate the body's neutral fat deposits and lipophilic receptor sites,and thereby increase the concentration of this type of allostericmodifiers in RBCs, where higher concentrations of effector will increaseits ability to interact with hemoglobin, causing delivery of moreoxygen. Ligands for the allosteric site of hemoglobin, also known asallosteric effectors of hemoglobin, include 2,3-diphosphoglycerate(DPG), inositol hexakisphosphate (IHP), bezafibrate (Bzf), LR16 and L35(two recently synthesized derivatives of Bzf), and pyridoxal phosphate.Additionally, hemoglobin's affinity for oxygen can be modulated throughelectrostatic interactions with chloride and/or organophosphate anionspresent in RBCs. These effectors, which bind preferentially to thedeoxy-Hb tetramers at a distance from the heme groups, play a major rolein the adaptation of the respiratory properties of hemoglobin to eitherallometric-dependent oxygen needs or to various hypoxic environments.Additionally, protons and carbon dioxide are physiological regulatorsfor the oxygen affinity of hemoglobin. The heterotropic allostericinteraction between the non-heme ligands and oxygen, collectively calledthe Bohr effect, facilitates not only the transport of oxygen but alsothe exchange of carbon dioxide.

The present invention relates to compositions, and methods of usethereof, consisting essentially of a nontoxic ammonia cation (preferablywater-soluble), and inositol hexaphosphate (IHP, phytic acid)derivatives comprising an internal pyrophosphate ring. IHP is the mostabundant form of phosphate in plants. IHP binds hemoglobin 1000 timesmore tightly than DPG and therefore triggers a decrease of theO₂/hemoglobin affinity with a subsequent release of oxygen. Because ofIHP's superior hemoglobin binding properties over DPG, IHP represents agood pharmaceutical candidate for diseases characterized by a limitedoxygen flow to organ tissues. Under normal physiological conditions, IHPbears at least 7 charges, making it very difficult for it to betransported across cell membranes. In order to answer the IHP deliveryproblem two approaches have been investigated: a) the ionic approach,which is based on a non-covalent interaction between IHP and thetransport molecules, and b) the prodrug approach, which is based on theidea that a linker covalently bound to IHP will facilitate the transportof the polyphosphate inside the red blood cells. Approach a) wasrealized with the synthesis of a library of IHP derivatives ionicallybound to lipophilic and non lipidic ammonium or polyammonium salts inU.S. application Ser. Nos. 09/920,310 and 09/920,140. The presentinvention expands upon approach b) wherein the covalently bound linkeris an adjacent phosphate group or an acyl phosphate group including acholesteryloxy carbonyl group, which under certain conditions eliminateto give an internal pyrophosphate ring.

In certain embodiments, the nontoxic ammonium cation is represented bythe general formula N⁺(R)₄, wherein R is, independently for eachoccurrence, H or an aliphatic group, which aliphatic group is preferablyan alkyl, preferably a lower (C1–C8) alkyl, and more preferably a C3–C10cyclic alkyl. In certain preferred embodiments, the ammonium cation ispreferably derived from cyclic organic bases. In a particularlypreferred embodiment, the ammonium cation isN,N-dimethylcyclohexylammonium (N,N-DMCHA) for the following reasons: a)it increases the lipophilisity of IHP and makes the molecule soluble inall organic solvents, without affecting its solubility in water, and b)as an ammonium salt of a tertiary amine, it doesn't react with the acylanhydrides or alkyl formates.

In certain embodiments, the present invention is related to compounds,and compositions thereof, which deliver IHP into erythrocytes in vivo,in vitro, or ex vivo. Additionally, the invention is directed to the useof the compounds or compositions thereof that are effective indelivering IHP into erythrocytes, lowering the oxygen affinity state inred blood cell suspensions and whole blood. It is an object of thisinvention to provide methods for delivering IHP into erythrocytes inwhole blood, utilizing compounds or compositions thereof that do notlose their effectiveness in the presence of normal concentrations of theremaining components of whole blood.

In certain embodiments, the present invention is related to a method oftreating red blood cells or whole blood in vivo, in vitro, or ex vivowith one or more nontoxic compounds or compositions of the presentinvention, suitably purifying said red blood cells or whole blood, andadministering said purified red blood cells or whole blood to a subjectfor any treatment where an increase in oxygen delivery by hemoglobinwould be a benefit.

In part, the present invention is directed toward the design ofwater-soluble membrane compatible molecules comprising ammonium cationicmoieties, e.g., lipophilic ammonium groups. These molecules formcomplexes with IHP derivatives comprising an internal pyrophosphatering; such complexes are useful for the delivery of IHP into thecytoplasm of erythrocytes. In the cases of the monopyrophospate andtripyrophosphate derivatives and acylated derivatives of IHP, metalcations, e.g., sodium cations, may allow deliver of IHP into thecytoplasm of erythrocytes.

The ammonium group of the cationic component of the compounds of thepresent invention is particularly well suited for interaction with thephosphate residues of IHP and congeners thereof because of the coulombicinteractions, i.e., the attraction between opposite charges, that can beestablished between the two moieties. The use of ammonium salts for theefficient delivery of IHP into mammalian erythrocytes is reported. Ourdata demonstrate the usefulness, convenience, and versatility ofammonium salts for delivery of IHP into the cytoplasm of mammaliancells.

In certain embodiments, the compounds of the present invention arerepresented by generalized structure I:

wherein

C⁺ represents independently for each occurrence an aliphatic ammoniumcation, an alkali metal cation, an alkaline earth cation, or othersuitable metal cation; and

A^(n−) represents a conjugate base of inositol hexaphosphate comprisingan internal pyrophosphate ring or an acyl group, wherein n equals thenumber of cations comprised by nC⁺.

In certain embodiments, the present invention relates to apharmaceutical composition, comprising a nontoxic compound of thepresent invention; and a pharmaceutically acceptable excipient.

IV. Preparation of IHP Derivatives Containing Internal PyrophosphateRings.

Our synthetic efforts toward acylated IHP derivatives was expected tohave competition from two side reactions. One was hydrolysis of the acylphosphates in water, a reaction dependent to some extent on pH. Theother competitive reaction was the formation of an internalpyrophosphate ring via elimination of the carbonyl adducts by a vicinalphosphate group. The latter reaction proved to be characteristic of theIHP derivatives.

In certain embodiments, IHP derivatives comprising an internalpyrophosphate ring were prepared by heating IHP with acyl anhydrides oracyl chlorides as depicted in Scheme 1.

The proposed mechanism for this reaction is depicted in Scheme 2. Thekey step is presumed to be conversion of a phosphate oxygen into aleaving group by forming an acyl phosphate ester. Once the acylphosphate ester forms, a vicinal phosphate group is well positioned toattack nucleophilically the central phosphorous atom and form theinternal pyrophosphate ring.

V. Synthesis of the IHP Tripyrophosphate (ITPP) Na Salt. Identificationof the Products of the Reactions of IHP with Excess of Acyl Anhydrides.

The literature procedures for the synthesis of the IHP tripyrophosphateinclude the conversion of the crystalline sodium phytate 1 to the freeacid 2, by passage through a column of Dowex 50 H⁺, Scheme 3. L. F.Johnson, M. E. Tate, Can. J. Chem., 1969, 47, 63. The column eluate wasadjusted to pH 8 with pyridine and evaporated to dryness to givecompound 3. The residue was dissolved in water and pyridine containingN,N-dicyclohexylcarbodiimide (DCC) was added. The reaction mixture washeated at 50–60° C. for 6 h and evaporated to dryness to give product 4(kf50A) as a pyridinium salt (checked by ¹H, ³¹P, ¹³C NMR-all phosphorusmoieties absorbed from −7 to −14 ppm at a pH range 2–3). The residue wasextracted with water, filtered and the filtrate adjusted to pH 10 with 5M NaOH. The sodium salt was precipitated by the addition of methanol andseparated by centrifugation to give product 5 (kf56) (checked by ¹H,³¹P, NMR-all phosphorus moieties absorbed from −7 to −14 ppm at a pH 9,see FIG. 7, spectrum 1a for the ³¹P NMR spectrum of compound 5 (kf56).

To further prove the synthesis of the tripyrophosphate sodium salt, partof compound 4 (kf50A) was passed through a Dowex Na⁺ exchange column andthe sodium salt 5 (kf53) was formed (Scheme 3, checked by ¹H, ³¹P,NMR-all phosphorus moieties absorbed from −7 to −14 ppm at a pH 6–7, seeFIG. 7, spectrum 1b for the ³¹P NMR spectrum of compound 5 (kf53)).

On the other hand, compound 6 (k122) has been synthesized from the IHPocta N,N-dimethylcyclohexylammonium salt 7 (kf36A), with excess ofbenzoic anhydride in refluxing acetonitrile for 24 h, while the Na saltwas derived from ion exchange of the mother compound after passingthrough a Dowex Na⁺ exchange resin column to give product 5 (kf31)(Scheme 3, checked by ¹H, ³¹P, NMR-all phosphorus moieties absorbed from−7 to −14 ppm at a pH 6–7, see FIG. 7, spectrum 1c for the ³¹P NMRspectrum of compound kf31). The pyrophosphate nature of the latterproduct 5 (kf31) was also revealed, when the pH was adjusted to 10 withaddition of 5 M NaOH (see FIG. 7, spectrum 1d for the ³¹P NMR spectrumof compound kf31A in pH 10).

Samples of all batches of compound 5 (kf31, kf53 and kf56) were mixedtogether and a mixed ³¹P NMR was run at pH 10, (see FIG. 8, spectrum2a). Both ¹H and ³¹P spectra of compounds kf31, kf53 and kf56 as well asof their mixture were found identical. The fact that the chemical shiftsof the compounds were insensitive to pH 6–10 suggests that all thephosphates were esterified.

Furthermore, a 2D ³¹P NMR COSY experiment of compound kf53 wasperformed, FIG. 8, spectrum 2b, showing a near classical pattern ofthree pairs of AB systems, with a strong correlation between thedoublets with centers at −8.40 and −13.19 ppm and J=21.2 Hz, and acorrelation between the doublets with centers at −9.58 and −9.71 ppm andJ=17.8 Hz. This exhibits another proof that pyrophosphates are presentin the molecule.

The synthesis of the pyrophosphates with DCC is explained with themechanistic scheme shown in Scheme 4, while the mechanistic scheme forthe reaction with acyl anhydrides and formates had been shown in Scheme2.

It is believed that the mechanism of Scheme 2 corresponding to formationof pyrophosphate upon reaction with acyl anhydrides under the conditionspresented herein is not presented in the literature. N. Li, R. F. Pratt,J. Am. Chem. Soc., 1998, 120, 4264–4268; M. Ahlmark, J. Versäläinen, H.Taipale, R. Niemi, T. Järvien, J. Med. Chem., 1999, 42, 1473–1476. Underdifferent conditions the synthesis of linear pyrophosphates to someextent was demonstrated, but such a formation was not observed here. H.G. Khorana, J. P. Vizsoyi, J. Am. Chem. Soc., 1959, 81, 4660. In oneexample, a 7 membered pyrophosphate ring of 1,4,5 myo-inositoltriphosphate was formed as a byproduct in a sequence of reactions whereacetic anhydride was involved. S. Ozaki, Y. Kondo, N. Shiotani, T.Ogasawara, Y. Watanabe, J. Chem. Soc. Perkin Trans. 1, 1992, 729–737. Inorder to investigate whether this was a trivial reaction in phosphatechemistry, DPG was exposed to the same conditions as with IHP, i.e.heating with an excess of acyl anhydride. Interestingly, no sign ofpyrophosphate CPPG was found in the reaction mixture. The differencebetween DPG and IHP in the behavior towards acyl anhydrides is that DPGcan have a free rotation around the bond connecting the two phosphatemoieties, Scheme 5. This allows the two highly charged groups to adopt aconformation where the two phosphates are far away from each, thuspromoting substitution rather than cyclization. Contrastly, in the caseof IHP, the six member carbocyclic ring forces the phosphates to stay ina close proximity. When a good leaving group is attached on one of thephosphates, the vicinal phosphate group attacks, and with elimination ofthe inserted group, the pyrophosphate is formed and the molecule is morestable energetically.

The orientation of IHP is such that pyrophosphates can form even withoutheating. In two control experiments, IHP octaN,N-dimethyl-cyclohexylammonium salt, 7 (kf36A), reacted with Ac₂O (6equiv in CH₂Cl₂ at rt for 4 days). The ³¹P NMR showed a very complexmixture due to the uncompleted reaction but the doublets of thepyrophosphates were clearly observed. The other control experimentconsisted of a reaction of the IHP octa N,N-dimethylcyclohexylammoniumsalt, 7 (kf36A), with DCC in CH₂Cl₂ at rt for 17 h. The reaction wasalso not completed but again the pyrophosphate doublets were detected.The same behavior of IHP was observed in the reactions with triphosgeneand formates.

VI. Synthesis of Inositol Tripyrophosphate (ITPP) Ammonium Salts fromPhytic Acid and ITPP Pyridinium Salt

The synthesis of ITPP derivatives through the two routes shown in Scheme6 were investigated. The first approach, Route A, led to the final ITPPcompound III following the pathway 1 to 2 to IV to III, while the secondone, Route B, was according to the pathway 1 to 2 to 4 to 8 to III.

Route A starts with compound 2, the perprotonated IHP molecule, andproceeds to the corresponding IHP compound IV. Compound IV bears themaximum of the counter cations they can hold. Compound IV was treatedwith DCC to give various results depending on the nature of the countercation. For example, in the case of the N,N-dimethylcyclohexylammoniumsalt of compound IV, the reaction goes to almost completion. In the caseof the n-hexylammonium-, cycloheptylammonium-, or cyclooctylammoniumsalts, 50% of the pyrophosphate product is hydrolyzed. The primary aminesalt solutions are strongly basic (pH>10) and it is believed that thehigh basicity causes hydrolysis of the initially formed pyrophosphates.To address this problem compound IV bearing 6 or less counter cationswere synthesized. Their reactions with DCC gave much better results, butnot the desirable pure compounds, (except in the case of the tertiaryammonium salt). Furthermore, this route was more strenuous because eachpyrophosphate had to be synthesized individually from its correspondingIHP ammonium salt in 3 steps. On the other hand, the more advancedcompound 4 having the pyrophosphates already formed, could be used as astarting material for the construction of desired compounds III.Additionally, 4 can be synthesized in large quantities and in very cleanform. These advantages prompted the investigation of alternative RouteB.

In Route B, the ITPP pyridinium salt 4 was synthesized according to theliterature procedure and is shown in detail in Scheme 3. L. F. Johnson,M. E. Tate, Can. J. Chem., 1969, 47, 63. The latter compound was passedthrough an ion exchange Dowex H⁺ to give the perprotonated ITPP compound8. Attempts to isolate this compound failed because pyrophosphates arevery vulnerable to acidic conditions and hydrolyze to IHP. Thus, when anaqueous solution of 8 was concentrated by evaporation and reacted withthe desired amine, part of the pyrophosphate was hydrolyzed givingmixtures of IHP and ITPP ammonium salts IV and III respectively.However, when the reaction was carried out immediately, with freshlyprepared and non isolated perprotonated 8, the ITPP salts III weresynthesized in excellent yields and in high purity.

General Route B has yielded 3 new ITPP salts of general structure III,bearing N,N-dimethylcyclohexylammonium (kf74), cycloheptylammonium(kf75) and cyclooctylammonium (kf76) counter cations (y=6). From theITPP pyridinium salt 4 (kf50A) the Na salt 5 (kf77) was prepared in theway shown in Scheme 3. All five compounds were initially examined invitro with free hemoglobin and whole blood.

VII. Solvent Effects on IHP-acyloxy Carbonyl Formation vs.IHP-pyrophosphate Formation.

According to the mechanism proposed in Scheme 2, the first step informing an internal pyrophosphate ring is acyl phosphoester formation.This mechanism also applies to compounds comprising an alkyloxy carbonylgroup. Experiments were carried out to determine what conditions favoredthe acyl or alkyloxy carbonyl intermediate versus internal pyrophosphatering formation. The effect of solvent on internal pyrophosphate ringformation was determined for CH₂Cl₂/1,4-dioxane, CH₃CN, and CH₃CN/THF.The effect of these solvents during purification was also investigated,as well as the stability of the acyl phosphoesters in water.

IHP octa N,N-dimethyl-cyclohexylammonium salt 7, reacted with 1 equiv ofRCOCl (R=cholesteryloxy) in a mixture of CH₂Cl₂ and 1,4-dioxane in aratio 2.5/1 for 5 days to give, as was identified by mass spectroscopy,the corresponding cholesteryloxycarbonyl derivative, Scheme 7.

In all cases the ³¹P-NMR spectra of the crude compounds were foundsimilar. However, they were all very complicated and could not beexplained as having a mixture of the 4 isomeric derivatives orcombinations of bis and/or mono cholesteryloxy carbonyl derivatives.FIG. 1 depicts a representative example of the ³¹P-NMR spectrum.

The spectra depict 3 bands of peaks. The first band covered the area 4to −1 ppm, that is the part of the spectrum where the orthophosphatesabsorb. The second band covered the area −4 to −9 ppm, and the third onecovered the area from −9 to −14 ppm. The last two bands were not foundin the ³¹P-NMR of the starting material (IHP) and therefore, belong tothe products of the reaction, meaning substituted phosphates. Allreactions, after purification, showed an increase in intensity of theband −9 to −14 ppm at the expense of the band −4 to −9 ppm. Thistransformation is likely of compounds that appear in the latter band(probably the cholesterol derivatives) to compounds that absorb in theformer band (internal pyrophosphate ring). Importantly, although 1equivalent of cholesteryl chloroformate was used as reagent, asignificant amount of cholesterol was always extracted during thepurification procedures.

In a first series of experiments, the same reaction conditions asreported above (1 eq CholCOCl, CH₂Cl₂/1,4-dioxane at r.t. for 5 days)were carried out, but purification was carried out under variousconditions, Table 1. Since there was pyrophosphate formed in thereaction, our task was at least to preserve the same amount ofpyrophosphate but not to increase it. For the purification of thereactions from cholesterol, we initially extracted with CH₂Cl₂/H₂O. Thesystem was forming a milky suspension and the two phases were not easilyseparated. At the end of the procedure, ³¹P-NMR showed that all peakshad moved into the −9 to −14 ppm area, indicating formation of theinternal pyrophosphate ring. It was decided to use another bi-phasesystem, hexanes/MeOH. Cholesterol is well soluble in hexanes and IHPcompounds are well soluble in MeOH. By heating at 50° C., the two phaseswere completely mixed in one phase. After cooling when the two phaseswere reseparated, the cholesterol had transferred into the hexanes phasemore efficiently. The result of this purification method, however, wasthe same as with CH₂Cl₂/1,4-dioxane.

More success was obtained with purifying the materials with non-proticsolvents, like hexanes, or hexanes in a mixture with some CH₂Cl₂ and/orcentrifugion of the mixtures, but still we were not able to retain theamount of the pyrophosphate at least at the initial portions.

TABLE 1 Synthesis of IHP Cholesteryloxy carbonyl derivatives inCH₂Cl₂/1,4-dioxane, and purification trials. pyrophosphate PyrophosphateSample Reaction before Purification after Name Conditions purificationConditions purification kf16 1 eq + Extraction +++ CholCOCl CH₂Cl₂/H₂OCH₂Cl₂/1,4- dioxane 2.5/1 (0.017 M to IHP salt) kf16a3 1 eq + Extraction+++ CholCOCl hexanes/MeOH CH₂Cl₂/1,4- heat 50° C., dioxane cool and2.5/1 separation (0.017 M to IHP salt) kf16.3 1 eq + Wash with hex, ++CholCOCl hex/CH₂Cl₂ CH₂Cl₂/1,4- dioxane 2.5/1 (0.017 M to IHP salt)kf16.4 1 eq + Wash with hex, ++ CholCOCl hex/CH₂Cl₂ CH₂Cl₂/1,4- 9/1dioxane 2.5/1 (0.017 M to IHP salt) kf49 1 eq +/+++ Wash with hex, ++++CholCOCl hex/CH₂Cl₂ CH₂Cl₂/1,4- 9/1 dioxane centrifugion 2.5/1 (0.017 Mto IHP salt) kf96 1 eq + Wash with hex, ++ CholCOCl hex/CH₂Cl₂CH₂Cl₂/1,4- 9/1 dioxane centrifugion 2.5/1 (0.017 M to IHP salt)

Interestingly, we found out, that samples kept in sealed vials-underair, were changing composition after a period of some weeks, giving morepyrophosphate.

In Table 2, we see the effect of the reaction time on the sidepyrophosphate reaction using our classical conditions, as well as asynthesis using a different solvent. It was obvious from reactions kf16,kf16a3, kf16.3, kf16.4, kf49, and kf96 that by increasing the reactiontime from 1 day to 3 days, the amount of the pyrophosphate was increasedas well. By changing the solvent from CH₂Cl₂/1,4-dioxane to CH₃CN (kf83)we realized for the first time a very clean reaction, althoughuncompleted in 22 h, because cholesteryl chloroformate was not very wellsoluble in CH₃CN.

TABLE 2 Synthesis of IHP Cholesteryloxy carbonyl derivatives undervarious conditions with 1 equivalent of cholesteryl chloroformate.Sample Name Reaction Conditions Pyrophosphate kf81A 1 eq CholCOCl +CH₂Cl₂/1,4-dioxane 2.5/1 (0.017 M to IHP salt) 1 day kf81B 1 eq CholCOCl++ CH₂Cl₂/1,4-dioxane 2.5/1 (0.017 M to IHP salt) 2 days kf81C 1 eqCholCOCl +++ CH₂Cl₂/1,4-dioxane 2.5/1 (0.017 M to IHP salt) 3 days kf831 eq. Chol. − CH₃CN, 22 h

However, by using a combination of THF, which dissolves the cholesterolreagent well, and CH₃CN, which dissolves the IHP starting material well,we were able to have again a clean reaction, kf88. Unfortunately,although kf88 was free of pyrophosphate, some was created during thepurification using the best conditions found at that moment. Theconcentration of the reaction in such dilute conditions is not importantsince kf91 also gave the same good results. As it was expected, thereaction time proved once again critical (kf99). See Table 3.

TABLE 3 Synthesis of IHP cholesteryloxycarbonyl derivatives inTHF/CH₃CN. Pyrophosphate Pyrophosphate Sample Reaction beforePurification after Name Conditions purification Conditions purificationkf88 1 eq of Chol − Washings with + THF:CH₃CN hexanes and 1:1 22 hhexanes/ (0.012 M CH₂Cl₂ 9/1, to IHP) centrifugation kf91 1 eq of Chol −none THF:CH₃CN 1:1 22 h (0.024 M to IHP) kf99 1 eq of Chol ++ noneTHF:CH₃CN 1:1 22 h 3 days

Finally, the results of changing the number of equivalents ofcholesteryl chloroform ate are presented in Table 4.

TABLE 4 Synthesis of IHP cholesteryl derivatives under THF/CH₃CNconditions with 1.5 and 2 equivalents of cholesterol chloroformate, andpurification trials. Pyrophosphate Pyrophosphate Sample Reaction beforePurification after Name Conditions purification conditions purificationkf89 1.5 eq Chol − Wash with + THF:CH₃CN hexanes, 1:1 22 h Hexanes/(0.012 M CH₂Cl₂ 9/1 IHP) centrifugation kf92 Same as − None kf89 (0.024M IHP) kf92p Same as − Wash with − kf92 hexanes, hexanes/THF 9/1Centrifugation kf100 Same as ++ None kf92 3 days kf90 2 eq Chol − Washwith + THF:CH₃CN hexanes, 1:1 22 h hexanes/ (0.012 M CH₂Cl₂ 9/1 IHP)centrifugation kf93 Same as − None kf90 (0.024 M IHP) kf93p Same as −Wash with − kf93 hexanes, hexanes/ THF 9/1 Centrifugation kf101 Same as++ None kf93

For reaction kf89 1.5 equiv of cholesteryl chloroform ate was used.According to the integration values of the ³¹P-NMR spectrum, in 22 hstatistically 1 cholesterol moiety was attached on IHP. The reaction wasnot forced to proceed for a longer period of time. Although theconcentration of the reaction mixture in these dilute conditions had noeffect (see reaction kf92), the increase of the reaction time was actingagainst the cholesterol derivatives (see reaction kf100). Reactionskf90, kf93, kf93p, and kf101 represent analogous reactions using 2 eq ofcholesteryl chloroformate.

When purification of kf89 and kf90 was attempted, pyrophosphates formedlike in case kf88. In all experiments using CH₂Cl₂, pyrophosphatesformed either in the reaction and/or in the purification. It is possiblethen, that CH₂Cl₂ accelerates the rate of pyrophosphate formation.Indeed, when THF was used in the purification step instead of CH₂Cl₂,pyrophosphate formation was avoided, and we were able to purify thealready pyrophosphate free products. The conclusion of the experimentsdescribed above is that alkyloxy carbonyl and acyl derivatives of IHPare relatively stable in organic solvents.

After completing the optimization and purification conditions, it wastime to check the behavior of the purified compounds in water. Based onthe stability that ATP cholesteryloxy carbonyl derivatives display inwater, it was assumed that the alkyloxy carbonyl IHP derivatives wouldbehave similarly. Because the biological applications of the presentinvention take place in water and in neutral pH, these parameters had tobe considered as well.

FIG. 2 depicts (a) the ³¹P NMR spectrum of the compound from kf96 inCDCl₃, and (b) the same purified compound in D₂O.

The difference observed in the absorption and the shape of the peaks wasdue to the D₂O. Interestingly, the spectrum of kf96 appeared lesscomplex in D₂O. The characteristic doublets of the pyrophosphates wereclearly detected, but the absence of peaks in the area at −4 to −8 ppmwas puzzling. The IHP monopyrophosphate was synthesized through areaction of IHP octa N,N-dimethyl-cyclohexylammonium salt 7 with 1equivalent of DCC (vide infra), and its spectrum is given in FIG. 3( c).Despite the fact that the spectra were complex due to the non selectiveformation of the pyrophosphates, they were all quite similar and closelyrelated to the ITPP N,N-dimethyl-cyclohexylammonium salt in D₂O (FIG. 3(d)), as it is shown from the comparison of spectra (a), (b) and (c) with(d). See FIG. 3.

Therefore, we concluded that IHP alkyloxy carbonyl derivatives are quiteunstable in water. They hydrolyse immediately either to pyrophosphatethrough an intramolecular attack from a neighboring phosphate, or toorthophosphate through an intermolecular attack from water. (The lattertransformation was hypothesized due to the loss of the 5:1 ratio of theintegration between orthophosphates and pyrophosphates in the spectra).This observation also explained, why prolonged storage in air waschanging the composition of the highly hygroscopiccholesteryloxycarbonyl compounds like kf49 in Table 1.

VIII. Synthesis of IHP Monopyrophosphate (IMPP)

Further experimentation led to control over the number of internalpyrophosphate rings formed. The synthesis of IHP-monopyrophosphate wascarried out using 1 equivalent of DCC (as activator of the phosphates)with 1 equivalent of IHP octa N,N-dimethyl-cyclohexyl ammonium salt in arefluxing mixture of CH₃CN/H₂O in a 2/1 ratio. See Scheme 8.

The synthesis is not selective, as it was revealed by the ³¹P NMR, seeFIG. 3( c), because all 6 phosphates of IHP have similar reactivity.Compound V passed through a Dowex Na⁺ exchange resin column to give thecorresponding Na⁺ salt. As will be seen in the following section,biologically active mixtures of the present compounds contain allpossible isomeric pyrophosphates if IHP. Therefore, tripyrophosphates ofIHP (ITPP) are advantageous because they are well defined compounds andcan be synthesized in high purity. ITPP compounds prepared thus far haveas their counterions N,N-dimethylcyclohexyl ammonium, pyridinium,cycloheptylammonium, cyclooctylammonium, or sodium.

IX. Detecting the Acyl-IHP Derivative Intermediate

The reactions of IHP with acyl anhydrides was investigated further inlight of both a) the optimization of the cholesteryloxy carbonyl IHPderivative synthesis and its behavior in both aqueous and organicsolutions, and b) the ability to control the competitive pyrophosphatereaction in organic solutions. A careful study of the spectra of earlierexperiments, revealed that acyl moieties do attach to IHP. However,before completely optimizing the reactions, their stability in aqueoussolutions and in neutral pH had to be examined.

The use of CH₂Cl₂ was avoided for reasons explained before. Instead, weexamined the reactions of IHP with 1–3 equivalents of Ac₂O, Bz₂O andhexanoic anhydride in either CH₃CN or a mixture of CH₃CN/THF. TheCH₃CN/THF solvent system was superior because it gave more loading ofacyls on IHP in the same amount of time. No pyrophosphates formed in allcases using these solvents.

A benzoate derivative, kf105, see FIG. 10, compound E for a generalstructure, synthesized upon a reaction of IHP octaN,N-dimethyl-cyclohexylammonium salt with 2 equiv of Bz₂O in a mixtureof CH₃CN/THF at r.t for 24 h, gave after concentrating the solvents acrude material. The ³¹P-NMR of kf105 in CDCl₃ is shown in FIG. 4( a).The absorption of the phosphorous peaks at −6 ppm indicates a benzoatesubstitution, since there was no phosphorous observed in the area of −8to −14 ppm. For the same crude material we performed a ³¹P-NMR spectrumin D₂O as shown in FIG. 4( b). Phosphorous absorption in the area around−5 ppm was retained which means the compound is stable in water.Finally, in order to prove that indeed we have a benzoate covalentlyattached on IHP, and that the formation of the pyrophosphate happensthrough an attack of a nearby phosphate according to mechanisms weproposed, we heated the material in CH₃CN for 6 h. As was expected, weobserved formation of pyrophosphates (area −9 to −14 ppm, FIG. 4( c)).The latter experiment showed that this phosphate benzoate mixedanhydride was quite stable, since after 6 h of heating itstransformation to the pyrophosphate was not thoroughly completed.

The next task was to determine if these compounds could tolerateconcentration in water and an increase in pH. To investigate theseparameters, another pilot experiment kf104, was carried out as follows:IHP octa N,N-dimethyl-cyclohexylammonium salt was reacted with 1 equivof Bz₂O in a mixture of CH₃CN/THF at r.t for 24 h. This reaction gaveafter concentration of the solvents a crude material, the ³¹P-NMR ofwhich in CDCl₃ is shown in FIG. 5( a). Comparing the spectra in FIG. 4(a) and FIG. 5( a) we see that using 1 equiv of Bz₂O had significantlyless loading of the benzoate (only approximately 20% of the reagent hadreacted in 24 h). The product was purified by extracting several timeswith toluene and H₂O in order to remove excess reagent. The aqueousphase was concentrated by rotary evaporation at 45° C. The ³¹P-NMRspectrum of the compound in CDCl₃ is depicted in FIG. 5( b). There wasno obvious change and the benzoate was still attached.

This material was redissolved in water and the pH of the solution wasadjusted to 6.9 with 0.2M NaOH. The mixture was concentrated as beforeand this time the ³¹P-NMR showed the characteristic doublets of thepyrophosphate and 25% hydrolysis. See FIG. 5 c. Hydrolysis to such asmall extent under these harsh conditions shows that it is quitepossible to prepare and study these prodrug anhydride derivatives.

An n-hexanoyl derivative, see FIG. 10, compound E for a generalstructure, was prepared by reacting IHP octaN,N-dimethyl-cyclohexylammonium with 2 equiv of (C₅H₁₁CO)₂O in CH₃CN atr.t for 24 h. The ³¹P-NMR in CDCl₃ is shown in FIG. 6( a). The hexanoylanhydride gave a higher loading than the benzoic anhydride (1 hexanoylis statistically attached on IHP, according to the integration of thespectrum). The mixture was extracted with toluene and water and theaqueous phase was concentrated as before. In FIG. 6( b), we see that thephosphorous peaks cover a larger area of the spectrum, but still wedon't see any evidence of pyrophosphate formation. It is believed thatthe broader coverage by the phosphorous peaks is due to migration of thehexanoyl moieties. Migration of acyl moieties is known in mediums ofacidic pH, and probably leads to a less strained and more stableconformation for the molecule.

The material was redissolved in water and a solution of 0.2M NaOH wasadded slowly at 0° C. (not at r.t.) until the solution arrived atpH=7.3. The solution was not concentrated but was instead lyophilized.The ³¹P-NMR of the compound (still soluble in CDCl₃) is depicted in FIG.6( c). No change was observed. The compound survived and remained intactthroughout all manipulations. Additionally, it did so at the correct pHfor the biological experiments.

In order to be sure that the distribution of the phosphates in thespectrum was not due to IHP transformations, the n-hexanoyl derivativewas heated in refluxing CH₃CN for 6 hours. It is clear from the ³¹P-NMRspectrum, (see FIG. 6( d)), that the acylated peaks disappeared and gaverise to pyrophosphate peaks. The reaction did not go to completion,showing that, as with the benzoate derivative, the phosphate acylanhydrides are indeed quite stable compounds. This has been repeatedlydemonstrated in the literature. N. Li, R. F. Pratt, J. Am. Chem. Soc.,1998, 120, 4264–4268; M. Ahlmark, J. Versäläinen, H. Taipale, R. Niemi,T. Järvien, J. Med. Chem., 1999, 42, 1473–1476.

X Attempts for Further Derivatization of ITPP

The ability of ITPP to react further with acylating or other agents wasinvestigated through three preliminary experiments. First, ITPPN,N-dimethylcyclohexyl ammonium salt was heated extensively with acylanhydrides and no change was observed. The second experiment attemptedto react ITPP pyridinium salt 9 with acyl anhydride in the presence ofpyridine and DMAP, but solubility of the starting material in CH₂Cl₂proved problematic. In the third experiment, a reaction with triphosgene(which was expected to exchange an OH with Cl) and subsequent reactionwith cycloheptyl amine gave, after extraction of the reaction mixturewith H₂O and CH₂Cl₂ separately, phosphorous containing compounds in bothphases (TLC, NMR). In the aqueous phase the ITPP cycloheptyl ammoniumsalt (instead of the pyridinium salt) was observed and in the organicphase a material absorbing in +10 ppm was observed which may be a fullysubstituted ITPP. Unfortunately this material is not soluble at all inwater and therefore could not serve as a prodrug.

From these preliminary experiments it was concluded that thepyrophosphate's free hydroxyl group is inactivate but not inert. It ispossible using the proper reagents to make ITPP even more lipophilicupon controlled substitutions and investigate the possibility oftransporting these molecules into the erythrocytes.

XI. Hydrolysis of Pyrophosphates.

Experiments designed to test IHP-pyrophosphate's resistance to chemicalhydrolysis were conducted. ITPP N,N-dimethyl-cyclohexylammonium saltsolutions were adjusted at pH 8.66, 10.20, 12.05 and 13.30. In order toavoid any damage to the NMR tubes due to the highly alkaline solutions,all samples were checked using ³¹P-NMR at different time intervalsagainst an external Ph₃PO/DMSO solution. This method also avoidedconcentrating the samples and redissolving them in a deuterated solvent,which may have led to changes in the results. No changes were observedafter 3 days, except for pH=13.3 which showed 7% hydrolysis. Observingthis stability, we exposed the pH=13.30 sample to heat at 60° C. for 6h. The sample revealed a non selective hydrolysis of only 13%. After 22h at the same temperature, hydrolysis was only 19%. Facing this extremestability to chemical hydrolysis we concentrated to dryness bothsolutions of pH 10.20 and 13.30 and dissolved them in D₂O. Their NMRspectra showed that the sample at pH 10.20 remained unchanged, while thepH=13.30 sample completely converted to the IHP open form sodium salt,phytic acid.

XII. Enzymatic Hydrolysis of ITPP

ITPP was dissolved in a buffer solution of a pH 4.6 and heated in thepresence of baker's yeast for 12 h at 45° C. Non selective hydrolysis ofapproximately 25% occurred.

XIII. Partition Coefficients of Pyrophosphates

Partition coefficients relate to the distribution of a solute betweentwo immiscible liquid phases and are defined as the ratios ofconcentrations (or molar fraction) of the distributed solute. These datahave been used to predict and rationalize numerous drug properties suchas quantitative structure/activity relationship, lipophilicity, andpharmacokinetic characteristics. 1-Octanol has been found to properlymimic biological membranes, and it has been estimated that1-octanol/water (K_(ow)) partition coefficients of more than 18000substances are now available in the literature.

The partition coefficients for our compounds,K_(ow)=[ITPP]_(1-octanol)/[ITPP]_(water), were measured afterequilibration at a concentration of 30 mM, close to the typicalconcentration employed for biological evaluations.

The IMPP hexa N,N-dimethyl-cyclohexylammonium compound and ITPPcompounds where the cation is pyridinium,N,N-dimethyl-cyclohexylammonium, and Na⁺ had K_(ow)<10⁻³ and could notbe measured using this method. Interestingly the cycloheptylammoniumITPP salt had K_(ow)=0.0121 and the cyclooctylammonium ITPP salt hadK_(ow)=0.462. This behavior is in agreement with what has been observedfor IHP cycloheptyl and cyclooctyl salts for their potential ability totransfer myo-inositol through cell membranes.

XIV. Partition Coefficients of Tripyrophosphate Na Salt as a Function ofa Cyclooctylammonium Concentration

It has previously been reported that cyclooctylammonium ions cantransport phytic acid into an octanol phase. S. P. Vincent, Jean-MarieLehn, J. Lazarte, C. Nicolau, Bioorg. Med. Chem., 2002, 10, 2825–2834.See FIG. 9. At a constant concentration (22 mM) 8 equiv of cyclohexylammonium ions are required to reach a K_(os) (in serum) value of 1,corresponding to an identical distribution between human serum and1-octanol. Similar results were obtained with the Na salt of ITPP in awater/1-octanol system. Considering that PP values in serum weregenerally lower that the ones in water, it was concluded that theincreased lipophilisity in ITPP (6 charges less than IHP) affects thetransportation of the compound with cyclooctylammonium salts. This meansmore equivalents of cyclooctylammoniums are needed in order to arrive ata K_(ow) of 1. This property indicates the significant differencebetween the two compounds in terms of physical behaviour.

XV. Oral Administration of Tri-pyrophosphates

The sodium salt of the tri-pyrophosphate derivative of IHP (kf111) wasdissolved in drinking (not deionized) water at a 20 g/L-concentration(=27 mM) and offered for drinking ad libitum. As in all experimentsperformed before, pH was adjusted to ˜7.0.

Twelve C57BL/6 mice drank kf111 over 4 days (about 25 ml/24 hrs). Threecontrol mice drank either pure water, or a solution of IHP (inositolhexaphosphate) at the same concentration and pH as kf111 (4 mice). Theamount of drunken fluid was the same when offering pure water, IHP-wateror kf111-water, indicating that kf111-, or IHP-solution was not rejectedby the mice. Blood was collected from the tail vein of the 19 C57BL/6mice on day 0 (before treatment started), 1, 2, 4, 6, 7, 8, 10, 11 and12, in order to measure P₅₀ values.

The following remarks can be made:

-   -   1. kf111 was not rejected by the mice apparently, when        administered orally.    -   2. kf111 was not harmful to the animals when applied orally. No        C57BL/6 mouse seemed to suffer by this treatment.    -   3. Oral application of kf111 caused significant right shifts of        P₅₀ (Up to 31%) in mice.

As described, the 19 C57BL/6 mice having received kf111 in water, IHP inwater or pure water were observed over 12 days, the P₅₀ values of theircirculating RBC were measured almost daily. FIG. 14 shows the timecourse of the induced right shift of the ODC (oxyhemoglobin dissociationcurve) in the mice ingesting kf111 and the absence of shift in thecontrol animals ingesting an aqueous solution of IHP or pure water.

It appears that all mice ingesting the aqueous solution of kf111 presenta shift of the P₅₀ value of their circulating RBC, albeit withindividual differences. None of the controls show a significant P₅₀shift. FIG. 2 illustrates the individual differences in the P₅₀ shiftinduced in the mice by ingestion of the aqueous solution of kf111.

XVI. Blood Counts of kf111—Treated and Control Mice

Blood from mice, having ingested kf111 or IHP in water (for 4 days) orwater only was collected on day 0, 7 and 11, in order to assess anydifferences in the blood count (and the amount of erythropoietin in thesera) of treated and control mice. Two major observations were made: 1.)The number of RBC in mice having ingested kf111 was reducedsignificantly, and 2.) There were no major differences in the number ofwhite blood cells (e.g. granulocytes, macrophages ect.) in blood frommice in different groups. Table 1 shows the RBC counts for mice withshifted ODC as compared to controls. Erythropoietin assays in all micesera will be reported soon.

TABLE 5 Number of RBC and P₅₀ shifts of treated and control animalsdetermined on days 7 and 10 of the experiment P₅₀ RBC x P₅₀ RBC x kf1117d % 10⁶/mm3 10d % 10⁶/mm3 Mouse 1 7 7.70 8 8.73 Mouse 3 16 6.54 11 7.65Mouse 4 9 6.54 9 7.80 Mouse 5 13 6.60 10 9.35 Mouse 6 14 5.73 6 8.60Mouse 7 20 6.35 10 8.95 Mouse 8 16 5.64 12 8.88 Mouse 11 15 5.45 10 8.95Mouse 12 20 8.76 16 8.70 Water 7 9.18 12 11.35 Water 4 8.7 1 10.95 IHP 39.6 0 10.77Values of 9 mice having received kf111, and 2 mice having received wateronly and 1 mouse having received IHP/water are shown. The amount ofblood from the other mice were not sufficient to determine the bloodcount. (On day 0 the RBC count in the mice was 8.9–11.8×10⁶ cells/mm³).Based on this data the following remarks can be made.

-   -   1.) kf111, when orally administered at a concentration of 27 mM,        causes a significant right shift of the P₅₀ value in murine        circulating RBC. There is a time lag of about 48 hrs before the        maximum shift is attained, contrarily to the observations made        after ip inoculation of kf111, where the P₅₀ shifts appears 2        hrs after inoculation.    -   2.) Maximal P₅₀ shifts are reached between day 2 and day 4 after        beginning oral administration of kf111.    -   3.) After 12 days P₅₀ values are back to control values (taken        on day 0), when ingestion is stopped on day 4.    -   4.) There is a significant effect of kf111 ingestion on the        number of RBC.    -   5.) The reason of this reduction has to be clarified: Hemolysis        of the RBC may be ruled out, as lysis of RBC never occurred in        vitro. The amount of erythropoietin in treated and control        animals will be reported soon.

It appears, that orally administered kf111 is effective in shifting theODC of circulating RBC in mice, even at modest concentrations of thecompound (27 mM).

XVII. Intravenous Injection of kf111 in Normal Pigs

An in vivo-experiment was performed on one 8 week-old normal piglet(body weight: 17 kg). The piglet was anaesthesized with 5% Isoflurane,0.7 L/min N₂O and 2.0 L/min O₂ for 20–30 minutes, when kf111 wasinjected, or blood was taken from the ear vein, respectively. Thecompound injected iv at a concentration of 27 g kf111/100 ml water(volume injected: 63 ml, pH 6.5, containing 17 g kf111=1 g/1 kg bodyweight) was not harmful to the animal, when injected into the piglet'sear vein over at least 10 minutes. The P₅₀ values of the porcine bloodobtained over 2 weeks after iv-injection are shown in FIG. 14.

XVIII. Blood Counts of the kf111 Treated Piglets

Blood from the 2 piglets, having received kf111 (1 g/kg body weight) wascollected before injection, 2 hrs after, and daily over a period of 14days after injection, in order to assess any differences in the bloodcounts of treated and non treated piglets. The following conclusions forpiglets having received 1 g kf111 per kg body weight can be drawn:

-   -   1. A slight decrease in hematocrit and in the number of RBC was        observed in the first days after injection.    -   2. A tendency towards the decrease of the reticulocytes (from        1.4% to 0.5%) was observed in blood samples collected the first        3 days after injection.    -   3. Increasing numbers of reticulocytes were counted in blood        samples of the injected animals taken 5–14 days after injection        (up to 3.0% on day 14).    -   4. Again, no major differences in the number of other cells,        such as white blood cells (e.g. granulocytes, macrophages,        platelets ect.) were detected.        XIX Dosis Effect Curve

Iv injection of 1 g kf111/kg body weight caused a significant rightshift of the P₅₀-value (up to 20%) in porcine RBCs. An almost saturatedkf111 solution, pH 6.7, was injected intravenously into two piglets(both of ˜18 kg body weight) (27 g kf111/100 ml=1.5 g/kg body weight)over 20 minutes.

Both piglets died, before the injection was completed (at that timepoint the animals had received <1.3 g/kg body weight=70–80 ml of thesaturated kf111-solution).

Blood was taken from the heart of the dead animals for the determinationof blood counts as well as the amount of sodium, potassium and calciumin the sera. All numbers of blood cells (hematocrit, white blood cellsect.) were halved. The amount of potassium and calcium was normal, whilesodium was doubled (before injection: 120–140 mmol/L; after injection:245 mmol/L). Apparently, the large amount of sodium in kf111 (6Na⁺/molecule) caused the death of the animals. It appears that up to 1 gkf111 per kg body weight can be injected iv, (if injected slowly)without harmful effects for the animals. The dosis effect curve is shownin FIG. 15. The following conclusions can be drawn from these results:

-   -   1. kf111 was not harmful to the piglet, when applied        intravenously slowly (at least 10 min for a vol. of solution of        100 ml)) at a concentration 1 g/kg* body weight. The piglets        were thirsty after the treatment.    -   2. Higher amounts of kf111, injected via iv, killed the animals.    -   3. A 1 g kf111 per kg body weight-injection is necessary to        cause a significant right shift of the P₅₀ value (up to 20%).    -   4. Pigs having received this amount of kf111, at that        concentration, did not show any pathological changes of the        blood counts, when injected slowly.    -   5. In piglets having received 1 g of kf111/kg body weight, a        tendency to the decrease in hematocrit was observed.    -   6. No major differences in the number of white blood cells (e.g.        granulocytes, macrophages, platelets ect.) in blood from the        treated piglets were detectable.    -   7. The number of reticulocytes decreased slightly 24 to 72 hrs        after injection (from 1.5% to 0.5%). Starting with day 3 after        injection of the allosteric effector, the number of        reticulocytes increased by about 3% for a period of 14 days. * a        second piglets was injected with kf111 at this concentration,        after 2 piglets had died after iv injection of 1.2 g kf111 (or        even more) per kg body weight.

EXEMPLIFICATION

Preparation of Effectors

The different synthetic pathways followed during our studies on IHPmolecules are briefly described in the general scheme for the synthesisof IHP derivatives. See FIG. 10.

Starting from Compound A, Route 1, upon reaction with excess of acylanhydrides (RCO)₂O(R═CH₃-kf12, kf20, C₂H₅-kf47, C₃H₇-kf43, C₄H₉-kf40,C₅H₁₁-kf46, C₆H₁₃-kf28, C₆H₅-kf13, kf22, CH₂═C(CH₃)-kf34 in CH₃CN reflux(see experimental section at the end of this review) we were targeting afully substituted hexa-acylated IHP that was thought to increase thelipophilisity of IHP by decreasing its charges by 6. The chemical shiftof the phosphorous were resonated around 10 ppm higher field than thestarting material. This fact was thought due to the shielding effect ofthe carboxylic substituent. However, instead of the expected products,and despite the fact that acyl anhydrides were described as stablecompounds in acidic to neutral pH and under elevated temperatures,another product was formed through dehydration of IHP. This productproved to be Compound B, the tripyrophosphate of IHP (ITPP), which hasalso 6 charges less than the starting material.

The same Compound B can be synthesized from Compound C, phytic acid,Route 2, upon reaction with DCC. Route 2, was described in theliterature—the products of Route 1 and 2 were found identical—and it wasthought more preferable for the synthesis of tripyrophosphates (than theone with the acyl anhydrides) due to the inexpense of the reagents (seeexperimental section). Furthermore, starting from the pyridinium salt ofITPP that can be synthesized in large quantities and in a clean form, wedeveloped a methodology to exchange the counter cations and createlibraries of ITPP ionically bound to lipophilic ammoniums (see exp.sect.). These salts exhibited interesting physicochemical and biologicalproperties. Especially the Na ITPP salt kf111 was the molecule with thebest profile, showing no toxicity even in a concentration of 160 mM. Theactivity of the ITPP molecules was predicted from the discovery of thefate of the cholesteryloxy carbonyl derivative in aqueous solutions,Route 3.

Reaction of Compound A, Route 3, with CholCOCl, (see experimentalsection) gave the Cholesteryloxy carbonyl derivative-kf16, kf38, kf42,kf96, which was stable only in some organic solvents, and for limitedperiod of time. Addition of water caused extensive hydrolysis back tothe starting material, as well as the formation of a new compound,IHP-monopyrophosphate (“IMPP”), Compound D. IMPP was also formed afterprolonged reaction times of compound A with CholCOCl in CH₂Cl₂.Therefore, all reactions made using the early procedures contained anabundant amount of IMPP. The remaining of the Chol derivative wasconverted to IMPP after addition of water—necessary for the biologicalexperiments—. The same Compound D can be synthesized upon reaction ofCompound A, with 1 equiv DCC, Route 4 (see exp. sect., kf109). ThisRoute was also found more preferable due to the inexpense of thereagents and the stoichiometry of the reaction. Under DCC conditionsonly IMPP is formed and we run no risk of hydrolysis from water andformation of the starting material A, (like in the case of Cholderivative). Furthermore, IMPP D, can be synthesized either from A, orC-phytic acid-kf149. The exchange of the counter cations is at the sametime possible and desired for the formation of more derivatives. The Nasalt kf133, kf152 again was found non-toxic as compared with theN,N-DMCHA salt kf109.

The formation of the IMPP is not selective, due to the similarreactivity of all the phosphate moieties of IHP. Since all possible IHPmonopyrophosphate isomers exist in the active pool of compounds, theircombination literally led to the design of the tripyrophosphate of IHPB, as a more promising candidate. The molecule being an internalanhydride of IHP showed no toxicity and excellent tolerability in the invivo biological experiments, especially with Na as counter cations,kf111. These compounds were easily synthesized as explained before usingRoute 2.

Finally, taking advantage of the best conditions found for the synthesisof the Chol derivative, we applied them on the synthesis of acylderivatives of IHP, Route 5. Indeed reaction of 1–3 equiv of acylanhydrides with Compound A gave (see exp. sect.) Compounds E in verygood yields, R═C₅H₁₁-kf137, kf151, kf160, kf161. The latter products arerelatively stable in water but with careful treatment we were able toperform extractions, and adjust their pH to 7. Furthermore we couldchange the counter cations to Na. Kf157-R═C₅H₁₁, kf158-R═CH₃, pH═7,kf137-R═C₅H₁₁, kf105-R═C₆H₅, upon heating in CH₃CN gave C Route 6,providing more proof that indeed acyl phosphate mixed anhydrides havebeen formed.

Synthesis of tripyrophosphates from reactions with acyl-anhydrides.Route 1 N,N-Dimethyl cyclohexyl ammonium Salt of Myo-inositol1,6:2,3:4,5-Tripyrophosphate, Compound B

IHP-octa-N,N-Dimethyl cyclohexyl ammonium salt A (1 equiv) was dissolvedin CH₃CN (0.02 mM) and acyl anhydride (RCO)₂O (20–30 equiv) was added.The mixture was refluxed for 24 h. (R═CH₃-kf12, kf20, C₂H₅-kf47,C₃H₇-kf43, C₄H₉-kf40, C₅H₁₁-kf46, C₆H₁₃-kf28, C₆H₅-kf13, kf22,CH₂═C(CH₃)-kf34). (In case of R═CH₃ no solvent was used. The mixture washeated in neat acetic anhydride at 120° C. for 24 h). The reactionmixture was cooled at 0° C. and water and toluene was added and themixture was extracted several times with toluene. The aqueous phase wasconcentrated to dryness, and the product was dried in vacuum to giveN,N-Dimethyl cyclohexyl ammonium Salt of Myo-inositol1,6:2,3:4,5-Tripyrophosphate.

Hexasodium Salt of Myoinositol 1,6:2,3:4,5-Tripyrophosphate, Compound B

The products of Route 1 were passing through an ion exchange Dowex 50Wx8Na⁺ form column, and the elute was concentrated in vacuum to givehexasodium Salt of Myo-inositol 1,6:2,3:4,5-Tripyrophosphate.

Synthesis of tripyrophosphate kf111. Route 2 Hexasodium Salt ofMyo-inositol 1,6:2,3:4,5-Tripyrophosphate, Compound B

See Can J. Chem. 1969, 47, 63–73.

Crystalline sodium phytate C (4 g) was dissolved with sonication inwater (20 ml) and converted to the free acid by passage through a columnof Dowex 50x8-200 ion-exchange resign. The column eluate was adjusted topH 8 with pyridine and evaporated to dryness. The residue was dissolvedin water (30 ml) and pyridine (130 ml) containingN,N-dicyclohexylcarbodiimide (8 g) was added. The reaction mixture washeated to 65° C. for 18 h and evaporated to dryness. The residue wasextracted with water (4×10 ml) filtered and the filtrate was evaporatedto dryness to give the pentapyridinium Salt of Myo-inositol1,6:2,3:4,5-Tripyrophosphate (3.355 g, 77% yield). ³¹P-NMR (D₂O) δ:−8.83 & −13.53 (AB, J=22.3 Hz, 2P, ax-eq), −9.82 & −10.00 (AB, J=17.8Hz, 2P, eq-eq), −10.18 (AB as a singlet, 2P, eq-eq); ¹H-NMR (D₂O) δ:8.65 (d, J=5.6 Hz, 10H), 8.48 (dd, J=7.9, 7.9 Hz, 5H), 7.94 (dd, J=7.0,7.0 Hz, 10H), 5.00 (bd, J=6.8 Hz, 1H), 4.57 (ddd, J=9.6, 9.6, 5.5 Hz,1H), 4.43–4.36 (m, 2H), 4.30–4.18 (m, 2H ); ¹³C-NMR (D₂O) δ: 147.0,140.9, 127.3, 77.9 (t, J=6.8 Hz), 76.4–76.0 (m), 75.4–75.0 (m), 73.8 (t,J=6.8 Hz), 73.3 (bs), 72.8 (bs). The compound was then dissolved inwater (30 ml) and passed through a column Dowex 50Wx8 Na⁺ form. Thecolumn eluate was concentrated to dryness to give Hexasodium Salt ofMyo-inositol 1,6:2,3:4,5-Tripyrophosphate (2.25 g, 97%) and used forbiological experiments in 98.5% purity without any further purification.The impurity is unreacted starting material (or tripyrophosphatehydrolyzed back to starting material). ³¹P-NMR (D₂O) δ: −8.34 & −13.14(AB, J=21.7 Hz, 2P, ax-eq), −9.53 & −9.70 (AB, J=17.8 Hz, 2P, eq-eq),−9.92 (AB as a singlet, 2P, eq-eq); ¹H-NMR (D₂O) δ: 5.04 (bd, J=10.5 Hz,1H), 4.65–4.59 (m, 1H), 4.51–4.36 (m, 2H), 4.32–4.18 (m, 2H); ¹³C-NMR(D₂O) δ: 77.1–76.8 (m), 76.5–76.0 (m), 75.4–75.0 (m), 74.1–73.9 (m),73.7–73.2 (m), 73.2–72.5 (m).

Synthesis of libraries of ammonium salts of tripyrophosphates. Route 2Ammonium Salts of Myoinositol 1,6:2,3:4,5-Tripyrophosphate, Compound B

The pentapyridinium Salt of Myo-inositol 1,6:2,3:4,5-Tripyrophosphate(the synthesis of which described before-literature procedure) convertedto the free acid by passage through a column of Dowex 50x8-200ion-exchange resign. The column eluate without any concentration was putin a round bottom flask and 6 equiv of the desired amine was added. Themixture was stirred at rt for 20 min and the mixture was evaporated todryness to give ammonium Salts of Myo-inositol1,6:2,3:4,5-Tripyrophosphate.

N,N-dimethyl cyclohexyl ammonium derivative

³¹P-NMR (CDCl₃) δ: −9.79 (AB as a singlet, 2P, eq-eq) −10.18 & −10.63(AB, J=21.2 Hz, 2P, eq-eq), −10.63 & −12.66 (AB, J=25.6 Hz, 2P, ax-eq);¹H-NMR (CDCl₃) δ: 5.42 (d, J=11.2 Hz, 1H), 4.76–4.67 (m, 1H), 4.59 (ddd,J=9.9, 9.9, 5.4 Hz, 1H), 4.36 (bdd, J=8.3, 8.3 Hz, 1H), 4.31–4.21 (m,1H), 4.05 (dd, J=9.5, 2.8 Hz, 1H), 2.95 (bs, 6H), 2.77 (s, 6×3×2 H),2.04 (bs, 12H), 1.82 (bs, 12H), 1.60 (d, J=12.3 Hz, 6H), 1.28 (bs, 24H),1.04 (bs, 6H); ¹³C-NMR (CDCl₃) δ: 75.2–74.9 (m), 74.2–73.7 (m),72.8–72.3 (m), 64.6, 39.4, 26.3, 25.0, 24.7. ³¹P-NMR (D₂O) δ: −8.76 &−13.48 (AB, J=23.4 Hz, 2P, ax-eq), −9.82 (AB as a singlet, 2P, eq-eq),−10.09 (AB as a singlet, 2P, eq-eq); ¹H-NMR (D₂O) δ: 5.04 (d, J=10.7 Hz,1H), 4.57–4.46 (m, 1H), 4.45–4.32 (m, 2H), 4.31–4.12 (m, 2H), 3.05 (bt,J=11.3 Hz, 6H), 2.69 (s, 36H), 1.90 (d, J=10.0 Hz, 12H), 1.77 (d, J=12.6Hz, 12H), 1.54 (d, J=12.7 Hz, 6H), 1.42–0.91 (m, 30H)

Cycloheptyl ammonium salt

³¹P-NMR (D₂O) δ: −8.61 & −13.37 (AB, J=23.4 Hz, 2P, ax-eq), −9.72 &−9.76 (AB, J=10.6 Hz, 1H), 2P, eq-eq), −10.02 (AB as a singlet, 2P,eq-eq); ¹H-NMR (D₂O) δ: 5.02 (bd, J=10.4 Hz, 1H), 4.60–4.49 (m, 1H),4.45–4.32 (m, 2H), 4.30–4.18 (m, 2H), 3.31–3.28 (m, 6H), 1.90–1.20 (m,1.70–1.25 (m, 60); ¹³C-NMR (D20) δ: 52.6, 32.29, 27.14, 23.14.

Cyclooctyl ammonium salt

³¹P-NMR (D₂O) δ: −8.62 & −13.38 (AB, J=23.4 Hz, 2P, ax-eq), −9.72 &−9.76 (AB, J=17.8 Hz, 2P, eq-eq), −10.03 (AB as a singlet, 2P, eq-eq);¹H-NMR (D₂O) δ: 5.02 (bd, J=10.6 Hz, 1H), 4.60–4.48 (m, 1H), 4.46–4.35(m, 2H), 4.32-4.15 (m, 2H), 3.40–3.32 (m, 6H), 1.90–1.20 (m, 84H);¹³C-NMR (D₂O) δ: 51.8, 30.2, 25.9, 24.96, 22.87.

Synthesis of the IHP Cholesteryoxy Carbonyl Hepta N,N-DimethylCyclohexyl Ammonium Salt. Route 3 IHP Cholesteryoxy Carbonyl HeptaN,N-Dimethyl Cyclohexyl Ammonium Salt

Initial conditions: material contaminated with monopyrophosphates, kf16,kf38, kf42, kf96.

IHP-octa-N,N-Dimethyl cyclohexyl ammonium salt A (3.094 g, 1.8506 mmol,1 equiv) was dissolved in CH₂Cl₂ (76 ml) and 1,4 dioxane (30 ml), andCholCOCl (873 mg, 1.94313 mmol, 1.05 equiv) was added in one portion.The mixture was stirred at rt under argon for 5 days, and concentratedto dryness. For the purification of 1.5 g of crude materialapproximately 1.5 lt of hex and hex/CH₂Cl₂ 9/1 were used as follows: Thesolid was washed with the solvents and the supertants were removed. Theremaining solid was again washed, until no cholesterol was observed byTLC.

kf96 ³¹P-NMR (CDCl₃) δ: 3.00–−2.5 (m, global integration 5P), −5.55–−6.7& −7.8–−9.6 & −10.2–−12.1 & −13.5–−14.8 (multiplets, global integration1P); ¹H-NMR (CDCl₃) δ: 4.90–0.10 (multiplets). ³¹P-NMR (D₂O) at pH=7 δ:4.07, 3.64, 3.49, 2.97, 2.85, 2.61, 2.48, 2.37, 1.90 (all singlets,global integration 5P), −7.80–−8.45 (3 doublets, J=23.4, 22.3, 23.4 Hz),−8.74–−10.03 (single and doublets, J=16.7, 16.7, 17.8 Hz), −12.53 (d,J=23.4 Hz), −13.01 (d, J=22.2 Hz); ¹H-NMR (D₂O) δ: 4.5–3.5 (multiplets),3.1–1.0 (peaks corresponding to N,N-DMCHA salt.

Improved conditions: material not contaminated with monopyrophosphates,kf92.

IHP-octa-N,N-Dimethyl cyclohexyl ammonium salt A (400 mg, 0.24 mmol, 1equiv) was dissolved in CH₃CN (5 ml) and THF (5 ml), and CholCOCl (161mg, 0.36 mmol, 1.5 equiv) was added in one portion. The mixture wasstirred at rt under argon for 24 h and concentrated to dryness. For thepurification of the crude material approximately 1 lt of hex/THF 9/1were used. The solid was washed with the solvents, centrifuged and thesupertants were removed. The remaining solid was again washed, until nocholesterol was observed by TLC.

kf92 ³¹P-NMR (CDCl₃) δ: 1.76 & −0.03 (2 broad multiplets as singlets,global integration 5P), 6.53 (bs, 1P); ¹H-NMR (CDCl₃) δ: 5.31 (bs),4.90–4.15 (m), 3.71(bs) 3.03 (bs), 2.79 (bs), 2.4–0.5 (m). ³¹P-NMR (D₂O)δ: 2.12–0.05 (many singlets), −8.84 (d, J=21.5 Hz), 9.46 (d, J=17.8 Hz),−9.80–−10.00 (m), −13.18 (d, J=17.8 Hz); ¹H-NMR (D₂O) δ: 4.90–4.73 (m,2H), 4.49–4.38 (m), 4.30–4.20 (m), 3.06 (bs, 6H), 2.69 (s, 36H), 1.83(bs, 12H), 1.76 (bs, 12H), 1.54 (d, J=11.9 Hz, 6H), 1.36–1.00 (m, 30H).

When the compound was dissolved in water, monopyrophosphate was formedin all cases.

Synthesis of the IHP monopyrophosphate. Route 4 IHP monopyrophosphateN,N-Dimethyl cyclohexyl ammonium salt, Compound D

Procedure No.1 from IHP-octa- N,N-Dimethyl cyclohexyl ammonium salt,reaction kf109.

IHP-octa-N,N-Dimethyl cyclohexyl ammonium salt A (1.175 g, 0.7 mmol, 1equiv) was dissolved in CH₃CN (20 ml) and H₂O (10 ml), and DCC (146.5mg, 0.7 mmol, 1. equiv) was added in one portion. The mixture wasrefluxed overnight, cooled to rt, the solid was filtrated and thefiltrate was concentrated to dryness. The residue was dissolved in waterand filtrated again. The filtrate was concentrated to dryness to giveIHP monopyrophosphate N,N-Dimethyl cyclohexyl ammonium salt.

³¹P-NMR (CDCl₃) δ: 2.9–−1.0 (many singlets, global integration 5P),−8.50–−10.9 (m, 1.4H doublets present with J=24.5, 16.7 Hz),−12.90–−13.7 (m, a main doublet J=22.8 Hz, 0.2P); ¹H-NMR (CDCl₃) δ:5.5–3.0 (bm), 2.87 (bs, 6H), 2.71 (bs, 36H), 2.02 (s, 12H), 1.56 (d,J=12.1 Hz, 6H), 1.24 (bs, 24H), 1.04 (bs, 6H).

IHP monopyrophosphate pyridinium salt, Compound D

Procedure No. 2 from IHP-dodecasodium salt, based on Can. J. Chem. 1969,47, 63–73, reaction kf149.

Crystalline sodium phytate C (2 g) was dissolved with sonication inwater (10 ml) and converted to the free acid by passage through a columnof Dowex 50x8-200 ion-exchange resign. The column eluate was adjusted topH 8 with pyridine and evaporated to dryness. The residue was dissolvedin water (14 ml) and pyridine (56 ml) containingN,N-dicyclohexylcarbodiimide (438 mg, 1 equiv) was added. The reactionmixture was heated to 65° C. for 18 h and evaporated to dryness. Theresidue was extracted with water (4×10 ml) filtered and the filtrate wasevaporated to dryness to give the IHP monopyrophosphate pyridinium salt.

³¹P-NMR (D₂O) δ: 2.05–−0.02 (singlets), −8.95 (d, J=21.9 Hz),−9.42–−10.21 (m), −13.25 (d, J=22.3 Hz); ¹H-NMR (D₂O) δ: 8.60 (d, J=5.3Hz), 8.43 (dd, J=8.0, 8.0 Hz), 7.89 (dd, J=6.9, 6.9 Hz), 5.04 (d, J=10.4Hz), 4.79 (d, J=11.7 Hz), 4.38–4.22 (m), 4.20–4.02 (m).

IHP monopyrophosphate sodium salt, Compound D

Compound kf109 or kf149 was dissolved in water and passed through acolumn Dowex 50Wx8 Na⁺ form. The column eluate was concentrated todryness to give IHP monopyrophosphate hexasodium salt, kf133, kf152,respectively.

³¹P-NMR (D₂O) δ: 2.26–0.42 (many singlets, 5P), −8.34 & −12.89 (ABdoublet, J=22.6 Hz, 0.5P), −9.00–−9.98 (m, 1.2P); ¹H-NMR (D₂O) δ: 5.10(bd, J=10.4 Hz), 4.80 (bd, J=9.9 Hz), 4.55–4.32 (m), 4.25–4.09 (m).

Synthesis of the IHP acyl compounds. Route 5 IHP acyl N,N-Dimethylcyclohexyl ammonium salt, Compound E

IHP-octa-N,N-Dimethyl cyclohexyl ammonium salt A (1 equiv) was dissolvedin CH₃CN or CH₃CN/THF and (RCO)₂O (1–3 equiv) was added in one portion,(R═CH₃-kf158, C₅H₁₁-kf137, kf151, kf160, kf161, R═C₆H₅-kf105). Themixture was stirred at rt for 24 h and concentrated to dryness. Waterand toluene (both ice cold) were added and the mixture was extractedseveral times with toluene. The aqueous phase was centrifuged to removeas much toluene as possible, and cooled to 0° C. Ice cold NaOH 0.2M wasadded dropwise until pH 7. The sample then was lyophilized to give IHPacyl N,N-Dimethyl cyclohexyl ammonium salt. Samples heated in CH₃CN gavemonopyrophosphate ROUTE 6

kf159 Benzoyl-IHP Na Salt

³¹P-NMR (CDCl₃) crude δ: 2.88–0.10 (many singlets), −5.09 (main singlet)ratio 7.5:1

kf137 Hexanoy-IHP NN-DMCHA Salt

³¹P-NMR (CDCl₃) pH=7 δ: 2.96–0.48 (many singlets), −4.96 & −5.65 & −8.04(main singlets), −5.00–−7.81 (other smaller singlets) total integrationratio 5 non acylated: 1; ¹H-NMR (CDCl₃) δ: 5.01–4.25 (3 multiplets, 6H),2.87 (bs), 2.72(s), 2.6–2.2 (m), 2.07 (bs), 1.84 (bs), 1.6.8–1.62 (m),1.40–1.10 (m) 0.86 (m as a d); ¹³C-NMR (CDCl₃): 171.2–169.8 (m).

kf158a Acetyl-IHP N,N-DMCHA Salt

³¹P-NMR (CDCl₃) crude δ: 2.90–−0.14 (many singlets), −5.63–−8.90 (manysinglets) ratio 2:1.

HP acyl sodium salt, Compound E

(Procedure like before until centrifugion). The aqueous phase was thenpassed through a column Dowex 50Wx8 Na⁺ form. The column eluate wascooled to 0° C. Ice cold NaOH 0.2M was added dropwise until pH 7. Thesample then was lyophilized to give IHP acyl sodium salt. (R═CH₃-kf₁₅₈,C₅H₁₁-kf157).

kf157 Hexanoy-IHP Na Salt

³¹P-NMR (D₂O) pH=7 δ: 3.81–0.08 (many singlets, 4.6P), −6.46 (mainsinglet), −5.84–−7.32 (other smaller singlets) global integration 1, and5% hydrolysed to pyrophosphate; ¹H-NMR (D₂O) δ: 4.92–4.80 (m), 4.38–4.25(m), 4.17–4.01 (m), 3.51 (q, J=7.1 Hz), 2.40 (bt, J=7.5 Hz), 2.03 (t,J=7.5 Hz), 1.58–1.35 (m), 1.30–1.11 (m, 2H), 1.04 (t, J=6.9 Hz, 5H),¹³C-NMR (CDCl₃): 173.15 (dd as a t, J=9.4 Hz), 77.3 (m), 76.2 (m), 75.0(m), 73.9 (m), 73.4 (m), 57.4, 37.4, 34.7 (3 peaks), 30.9, 30.4 (2peaks), 25.4, 23.5, 21.7 (2 peaks), 16.7, 13.2 (after one month in thefreezer some hydrolysis has occurred).

kf 158 Acetyl-IHP Na Salt

³¹P-NMR (D₂O) pH=7 δ: 3.60–1.18 (many singlets), −613–−7.90 (manysinglets, main singlet −6.97), 18% hydrolysed to pyrophosphate; ¹³C-NMR(D₂O):170.86–170.45 (m).

Experiments

A. In Vitro Experiments Performed with Whole Blood from Human, Mouse,and Pig.

The effectors k196 and kf111 (60 mM) were tested for P₅₀ shifts in wholeblood of three species: human, mouse and pig. As usual, pH's for thecompound-solutions were adjusted to ˜7.0, osmolarities for bothsolutions were determined (325–373 mOsM) prior to effectors, and wholeblood volumes at 1:1 ratios were incubated. Following incubation, bloodcells were washed 3 times with Bis-Tris-buffer (no lysis of RBCs wasobserved). A summary of P₅₀ values for whole blood induced by theeffectors is presented in Table 5.

TABLE 5 P₅₀ values in whole blood after incubation with effectors kf96and kf92p in vitro*. P₅₀ P₅₀ P₅₀ mm Hg mm Hg mm Hg effector P₅₀ effectorP₅₀ Blood CONTROL kf96 Increase % kf111 Increase % Human 22.1 28 27 30.839 Pig 32.2 41.1 27 45.2 40 Mouse 36.7 43.9 20 47.4 29 *only one animal(human) for each substance.

In all blood samples a strong right shift in the Hb-O₂ dissociationcurve was observed. The shifts obtained with kf111 (up to 40%) were evenstronger than with kf96 (27%). This and the fact that kf111 is welltolerated by mice even at a concentration of 120 mM led to a study whereseveral concentrations of kf111 (30 mM–150 mM) were injectedintraperitoneally to a group of 10 C57B1/6-mice for each concentration.At the present time we are performing this study by taking blood samplesfrom injected mice at 2 hours, I day, 4 days, and 12 days afterinjection, in order to measure P₅₀ shifts in blood and to follow thedecrease of P₅₀-shifts over time.

The concentration of the electrolytes sodium, potassium and calcium willbe determined after injection, in order to investigate possible sideeffects.

B. Investigation of the Effects of Intraperitoneal Injections of theEffector kf111.

Blood from C57B1/6 mice collected 2 hrs and 1 day after injection of 45,60, 120 and 150 mM solutions of kf111 was measured for P₅₀-shifts asreported. P₅₀-values of each single sample are listed in Table 6.Effector kf111 was well tolerated even at concentrations of 150 mM. Noanimal died or seemed to suffer from the compound. There was a shift OfP₅₀ at all concentrations.

TABLE 6 P₅₀ values of circulating RBC after ip-injection of the effectorkf111. Effector P₅₀ P₅₀ Concentration Shift % Shift % kf111 2 h Mean +/−SD* 24 h Mean +/− SD*  45 mM 12 11.8 +/− 1.16 13 13.6 +/− 1.02 11 15 1314 10 12 13 14  60 mM 12 16.9 +/− 3.48 14 17.2 +/− 2.1  14 16 17 17 2120 20.5 19 120 mM 28 26.0 +/− 2.28 28 24.8 +/− 2.7  29 28 24 22 26 24 2322 150 mM 26 27.0 +/− 1.78 25 25.8 +/− 2.78 28 26 30 31 26 24 25 23 P₅₀values of blood from 5 animals each are listed; *SD = standarddeviation.

C. In Vitro Experiments with Effectors kf133 and kf137 Performed withPig Hemoglobin and Whole Blood.

Two further effectors, kf133 and kf137, were tested in vitro for P₅₀shifts with porcine hemoglobin and whole blood. The compounds were wellsoluble. As usual, pH was adjusted to ˜7.0 and effector solution (2.5mM) and hemoglobin (2.5 mM) were mixed at a 1:1 ratio. Whole blood andthe effector solution were mixed at iso-osmolarity at pH=7. P₅₀ valueswere measured as described in the previous experiments. A summary of P₅₀values of free hemoglobin and whole blood induced by kf133 and kf137effectors is presented in Table 7.

TABLE 7 P₅₀ values of porcine free hemoglobin and whole blood afterincubation with the effectors kf133 and kf137 in vitro. P₅₀ P₅₀ mm Hg MmHg P₅₀ Control P₅₀ Effector Control P₅₀ Increase whole P₅₀ Increase Namehemoglobin mm Hg % blood mm Hg % kf133 20.4 48.5 143 33.5 38.4 14.5 19.9kf137 20.4 45.1 120 33.5 51.5 53.5 19.9The following remarks can be made regarding these measurements:

-   1. Both compounds caused a significant right shift with porcine    hemoglobin and whole blood. kf133 induced a P₅₀ shift of 143% with    hemoglobin.-   2. Whole blood incubated with kf133 gave a right shift of 14.5%.-   3. The new compound kf137 showed a right shift of 120% with free    porcine hemoglobin and surprisingly a very strong right shift with    porcine whole blood (of 53% and more) under approximately iso    osmolar conditions (288 mOsM).-   4. Noticeable lysis of the RBC was observed with effector kf137.

D. Investigation of the Effects of Intraperitonel Injection of theEffectors kf133 and kf137.

In order to evaluate the tolerability of the compounds kf133 and kf137,both were injected ip into C57B1/6 mice as described before. Again, thepH was adjusted to 6.8–7.2, and 200 μl of the solutions wereadministered ip. Intraperitoneal injection was well tolerated by mice ata concentration of up to 120 mM. P₅₀-shifts of circulating RBC were up20%.

Biological Evaluation

The biological evaluation of the effectors of Tables 1–4 are based inpart on results reported in U.S. provisional application 60/376,383,incorporated herein in its entirety by reference.

-   -   Effectors kf16.3, kf16.4, kf96, kf92p, and kf93p were shown to        cause a right shift in the P₅₀ value of both, free hemoglobin        and whole blood. The percentage of P₅₀ increase in hemoglobin        was up to 225%, in whole blood was up to 48%.    -   In vivo administration of 200 l of a 45 mM of kf16.3 solution        showed a right shift of the whole circulating blood of the        injected mice, shifts being up to 22% (demonstrating on 2        animals).    -   Administration of the kf16.4 compound 1p showed a significant        right shift in the P₅₀ of up to 18%, demonstrated on 6 animals).    -   The tolerability of the compound was tested by direct injection        into the vein at 30 and 45 mM: kf16 was non-toxic at these        concentrations.    -   The tolerability of the compound was tested also by        intraperitoneal injection at 30, 45 and 60 mM. kf16 was non        toxic at these 3 concentrations.    -   A significant right shift in the Hb—O₂ dissociation curve is        observed in vivo upon IP injection of the highly purified kf16.5        compound. This shift is concentration dependent. The injection        of 200 μl of a 30 mM—kf96 caused a shift of up to 17%, a 45        mM—solution a shift up to 22%, and a 60 mM-solution a shift up        to 24%.    -   The decrease of the P₅₀ shift over time is progressive and        correlates with the life-time of the mouse RBC. This indicates        that we have a true shift, induced by the allosteric effector        injected.    -   Significant right shifts of the ODC of circulating RBC could be        obtained in vivo also with IHP-pyrophosphates, mainly using        IHP-tris-pyrophosphates.    -   The shifts amounted to 24% of the basal value, either when        incubated with RBC in vitro or after intraperitoneal injection.    -   When injected to a limited number of animals, “cholesterol”        derivatives were toxic at concentrations of 45 mM or upwards,        whereas tripyrophosphates appear to be non toxic at much higher        concentrations (60–120 mM).    -   Significant right shifts, 31% of the basal value after ip        injection, of the ODC of circulating RBC could be obtained in        vivo with effector kf111.    -   When injected up to 150 mM to a limited number of mice, effector        kf111 was well tolerated.    -   Effectors kf111 and kf96 were tested with human and porcine        blood: strong right shifts (up to 40%) were observed with        effector kf111 in blood of all three species.    -   The newly synthesized allosteric effector kf137 was tested in        vitro with porcine blood. A greater than 50% increase of the P₅₀        value was measured, which is the strongest shift in whole blood        we ever observed in vitro with any IHP-derivative. As soon as        sufficient amounts of purified effector is available,        experiments will be conducted with murine, human, porcine, and        canine blood in vitro.

CONCLUSION

Inositol pyrophosphates represent a new class of allosteric effectors ofhemoglobin. Their highly charged nature allows them to pass througherythrocyte membranes and bind to hemoglobin, resulting in a significantright P₅₀ value. The progressive decrease of the P₅₀ shift over timecorrelates with the life time of mouse red blood cells. This indicates atrue shift, induced by the inositol pyrophosphate allosteric effector.It is envisioned by the present invention that further derivatization ofITPP, IMPP, or the synthesis of more pyrophosphate containing compoundsmay lead to other allosteric effectors. The understanding of themechanism of the transportation of these IHP compounds throughoutmembranes is also expected to lead to compounds with interestingproperties.

Incorporation by Reference

All of the patents and publications cited herein are hereby incorporatedby reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areencompassed by the following claims.

1. A composition, comprising a salt complex comprising an aliphaticammonium cation, and an anionic ligand for an allosteric site ofhemoglobin, wherein the anionic ligand comprises an internalpyrophosphate ring.
 2. The composition of claim 1, wherein the anionicligand comprises two internal pyrophosphate rings.
 3. The composition ofclaim 2, wherein the anionic ligand comprises three internalpyrophosphate rings.
 4. The composition of claim 1, wherein the anionicligand is a phosphorylated inositol.
 5. The composition of claim 1,wherein the anionic ligand is an inositol hexaphosphate.
 6. Thecomposition of claim 1, wherein the aliphatic ammonium cation is alipophilic, water-soluble aliphatic ammonium cation.
 7. The compositionof claim 1, wherein the aliphatic ammonium cation is a monoalkyl,dialkyl, trialkyl or tetraalkyl ammonium moiety.
 8. The composition ofclaim 1, wherein the aliphatic ammonium cation is anN,N-dimethylcyclohexylammonium cation.
 9. The composition of claim 1,wherein the aliphatic ammonium cation is a monoalkyl ammonium cation.10. The composition of claim 1, wherein the aliphatic ammonium cation isa primary ammonium cation.
 11. A pharmaceutical formulation, comprisinga composition of claim 1; and a pharmaceutically acceptable excipient.12. A method of enhancing oxygen delivery to a tissue or organ of amammal, comprising the step of administering to a mammal atherapeutically effective amount of a composition of claim
 1. 13. Themethod of claim 12, wherein the composition is administered orally. 14.A method of enhancing oxygen delivery to a tissue or organ of a mammal,comprising the step of administering to a mammal red blood cellspreviously treated with a composition of claim 1.